Anti-IL31 Antibodies for Veterinary Use

ABSTRACT

Provided are various embodiments relating to anti-IL31 antibodies binding to canine IL31. Such antibodies can be used in methods to treat IL31-induced conditions in companion animals, such as canines, felines, and equines.

This application is a divisional of U.S. application Ser. No.16/186,013, filed Nov. 9, 2018, which is a divisional of U.S.application Ser. No. 15/844,142, filed Dec. 15, 2017, now U.S. Pat. No.10,150,810, which is a divisional of Ser. No. 15/467,464, filed Mar. 23,2017, now U.S. Pat. No. 10,093,731, which claims the benefit of U.S.Provisional Application No. 62/463,543, filed Feb. 24, 2017, each ofwhich is incorporated by reference herein in its entirety for anypurpose.

FIELD OF THE INVENTION

This invention relates to isolated anti-IL31 antibodies, for example,binding to canine IL31, and methods of using the same, for example,treating IL31-induced conditions or reducing IL31 signaling function incells, for instance in companion animals, such as canines, felines, andequines.

BACKGROUND

Interleukin 31 (IL31) is a cytokine mostly produced by Th2 cells andunderstood to be involved in promoting skin disease, such as pruriticand other forms of allergic diseases (for example, atopic dermatitis).IL31 functions by binding its receptor and activating downstreamactivities, such as activation of JAK1, and is thought to cause many ofthe clinical problems associated with dermatitis and other disorders.

Companion animals such as cats, dogs, and horses, suffer from many skindiseases similar to human skin diseases, including atopic dermatitis.However, the IL31 sequence is divergent between human, cat, dog, andhorse. There remains a need, therefore, for methods and compounds thatcan be used specifically to bind companion animal IL31 for treatingIL31-induced conditions and for reducing IL31 signaling.

SUMMARY OF THE INVENTION

In some embodiments, an isolated antibody is provided that binds tocanine IL31. In some embodiments the antibody binds to an epitopecomprising amino acids 34-50 of SEQ ID NO: 22. In some embodiments, theantibody binds to an epitope comprising the amino acid sequence of SEQID NO: 23.

In some embodiments, the antibody binds to canine IL31 with adissociation constant (Kd) of less than 5×10⁻⁶ M, less than 1×10⁻⁶ M,less than 5×10⁻⁷ M, less than 1×10⁻⁷ M, less than 5×10⁻⁸ M, less than1×10⁻⁸ M, less than 5×10⁻⁹ M, less than 1×10⁻⁹ M, less than 5×10⁻¹⁰ M,less than 1×10⁻¹⁰ M, less than 5×10⁻¹¹ M, less than 1×10⁻¹¹ M, less than5×10⁻¹² M, or less than 1×10⁻¹² M, as measured by biolayerinterferometry.

In some embodiments, the antibody reduces IL31 signaling function in acompanion animal species, as measured by a reduction in STAT-3phosphorylation. In some embodiments, the companion animal species iscanine, feline, or equine.

In some embodiments, the antibody binds to feline IL31 or equine IL31,as determined by immunoblot analysis or biolayer interferometry. In someembodiments, the antibody competes with monoclonal M14 antibody inbinding to canine IL31. In some embodiments, the antibody of any one ofclaims 1-5, wherein the antibody competes with monoclonal M14 antibodyin binding to feline IL31 or in binding to equine IL31.

In some embodiments, the antibody is a monoclonal antibody. In someembodiments, the antibody is a canine, a caninized, a feline, afelinized, an equine, an equinized, or a chimeric antibody. In someembodiments, the antibody is a chimeric antibody comprising murinevariable heavy chain framework regions or murine variable light chainframework regions.

In some embodiments, the antibody comprises a heavy chain and a lightchain, wherein:

a. the heavy chain comprises a CDR-H1 sequence having at least 85%sequence identity, at least 90% sequence identity, at least 95% sequenceidentity, or at least 98% sequence identity to the amino acid sequenceof SEQ ID NO: 1; a CDR-H2 sequence having at least 85% sequenceidentity, at least 90% sequence identity, at least 95% sequenceidentity, or at least 98% sequence identity to the amino acid sequenceof SEQ ID NO: 2; and a CDR-H3 sequence having at least 85% sequenceidentity, at least 90% sequence identity, at least 95% sequenceidentity, or at least 98% sequence identity to the amino acid sequenceof SEQ ID NO: 3, andb. the light chain comprises a CDR-L1 sequence having at least 85%sequence identity, at least 90% sequence identity, at least 95% sequenceidentity, or at least 98% sequence identity to the amino acid sequenceof SEQ ID NO: 8; a CDR-L2 sequence having at least 85% sequenceidentity, at least 90% sequence identity, at least 95% sequenceidentity, or at least 98% sequence identity to the amino acid sequenceof SEQ ID NO: 9; and a CDR-L3 sequence having at least 85% sequenceidentity, at least 90% sequence identity, at least 95% sequenceidentity, or at least 98% sequence identity to the amino acid sequenceof SEQ ID NO: 10.

In some embodiments, the antibody comprises a heavy chain comprising (a)a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (b) aCDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (c) aCDR-H3 comprising the amino acid sequence of SEQ ID NO: 3. In someembodiments, the antibody comprises a light chain comprising (a) aCDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, (b) a CDR-L2comprising the amino acid sequence of SEQ ID NO: 9, and (c) a CDR-L3comprising the amino acid sequence of SEQ ID NO: 10.

In some embodiments, the antibody comprises one or more of (a) avariable region heavy chain framework 1 (HC-FR1) sequence of SEQ ID NO:4, (b) a HC-FR2 sequence of SEQ ID NO: 5, (c) a HC-FR3 sequence of SEQID NO: 6, (d) a HC-FR4 sequence of SEQ ID NO: 7, (e) a variable regionlight chain framework 1 (LC-FR1) sequence of SEQ ID NO: 11, (f) anLC-FR2 sequence of SEQ ID NO: 12, (g) an LC-FR3 sequence of SEQ ID NO:13, or (h) an LC-FR4 sequence of SEQ ID NO: 14.

In some embodiments, the antibody comprises:

a. (i) a variable light chain sequence having at least 85%, at least90%, at least 95%, or at least 98% sequence identity to the amino acidsequence of SEQ ID NO: 24; (ii) a variable heavy chain sequence havingat least 85%, at least 90%, at least 95%, or at least 98% sequenceidentity to the amino acid sequence of SEQ ID NO: 25; or (iii) avariable light chain sequence as in (i) and a variable heavy chainsequence as in (ii); orb. (i) a variable light chain sequence having at least 85%, at least90%, at least 95%, or at least 98% sequence identity to the amino acidsequence of SEQ ID NO: 16; (ii) a variable heavy chain sequence havingat least 85%, at least 90%, at least 95%, or at least 98% sequenceidentity to the amino acid sequence of SEQ ID NO: 15; or (iii) avariable light chain sequence as in (i) and a variable heavy chainsequence as in (ii); orc. (i) a variable light chain sequence having at least 85%, at least90%, at least 95%, or at least 98% sequence identity to the amino acidsequence of SEQ ID NO: 32; (ii) a variable heavy chain sequence havingat least 85%, at least 90%, at least 95%, or at least 98% sequenceidentity to the amino acid sequence of SEQ ID NO: 33; or (iii) avariable light chain sequence as in (i) and a variable heavy chainsequence as in (ii).

In some embodiments, the antibody comprises a variable light chainsequence of SEQ ID NO: 24; SEQ ID NO: 16; or SEQ ID NO: 32. In someembodiments, the antibody comprises a variable heavy chain sequence SEQID NO: 25; SEQ ID NO: 15; or SEQ ID NO: 33. In some embodiments, theantibody comprises: a variable light chain sequence of SEQ ID NO: 24 anda variable heavy chain sequence of SEQ ID NO: 25; a variable light chainsequence of SEQ ID NO: 16 and a variable heavy chain sequence of SEQ IDNO: 15; or a variable light chain sequence of SEQ ID NO: 32 and avariable heavy chain sequence of SEQ ID NO: 33.

In some embodiments, the antibody is a chimeric antibody comprising aconstant heavy chain region or constant light chain region derived froma companion animal.

In some embodiments, the antibody comprises (a) a canine heavy chainconstant region selected from an IgG-A, IgG-B, IgG-C, and IgG-D constantregion; (b) a feline heavy chain constant region selected from an IgG1,IgG2a, and IgG2b constant region; or (c) an equine heavy chain constantregion selected from an IgG1, IgG2, IgG3, IgG4, IgG5, IgG6 and IgG7constant region.

In some embodiments, the antibody comprises:

a. (i) a light chain amino acid sequence of SEQ ID NO: 26; (ii) a heavychain amino acid sequence of SEQ ID NO: 27; or (iii) a light chain aminoacid sequence as in (i) and a heavy chain amino acid sequence as in(ii); orb. (i) a light chain amino acid sequence of SEQ ID NO: 30; (ii) a heavychain amino acid sequence of SEQ ID NO: 31; or (iii) a light chain aminoacid sequence as in (i) and a heavy chain amino acid sequence as in(ii); orc. (i) a light chain amino acid sequence of SEQ ID NO: 34; (ii) a heavychain amino acid sequence of SEQ ID NO: 35, or (iii) a light chain aminoacid sequence as in (i) and a heavy chain amino acid sequence as in(ii).

In some embodiments, the antibody comprises a light chain amino acidsequence of SEQ ID NO: 21. In some embodiments, the antibody comprises aheavy chain amino acid sequence of SEQ ID NO: 17; SEQ ID NO: 18; SEQ IDNO: 19; or SEQ ID NO: 20.

In some embodiments, the antibody is an antibody fragment selected fromFv, scFv, Fab, Fab′, F(ab′)2, and Fab′-SH.

In some embodiments, the antibody is bi-specific, wherein the antibodybinds to IL31 and one or more antigens selected from IL17, TNFα, CD20,CD19, CD25, IL4, IL13, IL23, IgE, CD11α, IL6R, α4-Intergrin, IL12, IL1β,or BlyS.

In some embodiments, an isolated nucleic acid is provided, which encodesan anti-IL31 antibody described herein above. In some embodiments, ahost cell is provided, which comprises a nucleic acid encoding ananti-IL31 antibody described herein above. In some embodiments, a methodof producing an anti-IL31 antibody is provided, which comprisesculturing such a host cell comprising a nucleic acid encoding ananti-IL31 antibody described herein above and isolating the antibody. Insome embodiments, a pharmaceutical composition is provided, whichcomprises an anti-IL31 antibody described herein and a pharmaceuticallyacceptable carrier.

In some embodiments, methods of treating a companion animal specieshaving an IL31-induced condition are provided, comprising administeringto the companion animal species a therapeutically effective amount of ananti-IL31 antibody described herein or a pharmaceutical compositioncomprising the antibody described herein. In some embodiments, thecompanion animal species is canine, feline, or equine. In someembodiments, the IL31-induced condition is a pruritic or allergiccondition. In some embodiments, the IL31-induced condition is selectedfrom atopic dermatitis, pruritus, asthma, psoriasis, scleroderma andeczema.

In some embodiments, the anti-IL31 antibody or the pharmaceuticalcomposition is administered parenterally. In some embodiments, theanti-IL31 antibody or the pharmaceutical composition is administered byan intramuscular route, an intraperitoneal route, an intracerebrospinalroute, a subcutaneous route, an intra-arterial route, an intrasynovialroute, an intrathecal route, or an inhalation route.

In some embodiments, the method comprises administering in combinationwith the anti-IL31 antibody or the pharmaceutical composition a Jakinhibitor, a PI3K inhibitor, an AKT inhibitor, or a MAPK inhibitor. Insome embodiments, the method comprises administering in combination withthe anti-IL31 antibody or the pharmaceutical composition one or moreantibodies selected from an anti-IL17 antibody, an anti-TNFα antibody,an anti-CD20 antibody, an anti-CD19 antibody, an anti-CD25 antibody, ananti-IL4 antibody, an anti-IL13 antibody, an anti-IL23 antibody, ananti-IgE antibody, an anti-CD11α antibody, anti-IL6R antibody,anti-α4-Intergrin antibody, an anti-IL12 antibody, an anti-IL1βantibody, and an anti-BlyS antibody.

In some embodiments, methods of reducing IL31 signaling function in acell are provided, comprising exposing to the cell an anti-IL31 antibodythe pharmaceutical composition described herein under conditionspermissive for binding of the antibody to extracellular IL31, therebyreducing binding to IL31 receptor and/or reducing IL31 signalingfunction by the cell. In some embodiments, the cell is exposed to theantibody or the pharmaceutical composition ex vivo. In some embodiments,the cell is exposed to the antibody or the pharmaceutical composition invivo. In some embodiments, the cell is a canine cell, a feline cell, oran equine cell.

In some embodiments, a method for detecting IL31 in a sample from acompanion animal species are provided, comprising contacting the samplewith an anti-IL31 antibody or the pharmaceutical composition describedherein under conditions permissive for binding of the antibody to IL31,and detecting whether a complex is formed between the antibody and IL31in the sample. In some embodiments, the sample is a biological sampleobtained from a canine, a feline, or an equine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is an alignment of variable light sequences of M14, M18, M19,and M87 mouse monoclonal antibody clones. FIG. 1B is an alignment ofvariable heavy sequences of M14, M18, M19, and M87 mouse monoclonalantibody clones.

FIG. 2A and FIG. 2B are graphs of canine IL31 binding analysis withvarying concentrations of chimeric M14 antibody.

FIG. 3A and FIG. 3B are graphs of canine IL31 binding analysis withvarying concentrations of caninized M14 antibody.

FIG. 4 is an immunoblot showing inhibited canine IL31 signaling atvarying concentrations of caninized M14 antibody.

FIGS. 5A and 5B are immunoblots of GST-canine-IL31 deletions probed withM14 antibody and anti-GST antibody, respectively.

FIGS. 6A and 6B are immunoblots of GST-canine-IL31 deletions probed withM14 antibody and anti-GST antibody, respectively.

FIGS. 7A and 7B are immunoblots of feline and equine IL31 proteins fusedto human Fc probed with M14 antibody and anti-FC antibody, respectively.

DESCRIPTION OF CERTAIN SEQUENCES

TABLE 1Table 1 provides a listing of certain sequences referenced herein.Description of Certain Sequences SEQ ID NO: SEQUENCE DESCRIPTION  1GDSITSGYW Variable heavy chain CDR-H1 amino acid sequence of mouseantibody clone M14  2 YISYSGITDYNPSLKS Variable heavy chain CDR-H2amino acid sequence of mouse antibody clone M14  3 ARYGNYGYAMDYVariable heavy chain CDR-H3 amino acid sequence of mouseantibody clone M14  4 EVQLQESGPSLVKPSQTLSLTCSVTVariable region heavy chain framework HC-FR1 aminoacid sequence of mouse antibody clone M14  5 NWIRKFPGNKLEYMGVariable region heavy chain framework HC-FR2 aminoacid sequence of mouse antibody clone M14  6RISITRDTSKNQYYLQLNSVTTEDTATYYC Variable region heavy chainframework HC-FR3 amino acid sequence of mouse antibody clone M14  7WGQGTSVTVSS Variable region heavy chain framework HC-FR4 aminoacid sequence of mouse antibody clone M14  8 RASESVDTYGNSFMHVariable light chain CDR-L1 amino acid sequence of mouseantibody clone M14  9 RASNLES Variable light chain CDR-L2amino acid sequence of mouse antibody clone M14 10 QQSYEDPWTVariable light chain CDR-L3 amino acid sequence of mouseantibody clone M14 11 DIVLTQSPASLAVSLGQRATISCVariable region light chain framework LC-FR1 aminoacid sequence of mouse antibody clone M14 12 WYQQKSGQSPKLLIYVariable region light chain framework LC-FR2 aminoacid sequence of mouse antibody clone M14 13GIPARFGGSGSRTDFTLTIDPVEADDVATYYC Variable region light chainframework LC-FR3 amino acid sequence of mouse antibody clone M14 14FGGGTKLEIK Variable region light chain framework LC-FR4 aminoacid sequence of mouse antibody clone M14 15EVQLVESGPSLVKPGGSLRLTCSVTGDSITSGYWNWI Caninized variable heavy chainRKFPGNKLEYMGYISYSGITDYNPSLKSRITISRDTS amino acid sequence of mouseKNQYYLQLNSVTTEDTATYYCARYGNYGYAMDYWGQG antibody clone M14 TLVTVSS 16DIVMTQSPASLSVSLGQRATISCRASESVDTYGNSFM Caninized variable light chainHWYQQKPGQSPKLLIYRASNLESGIPARFGGSGSGTD amino acid sequence of mouseFTLTIDPVQADDVATYYCQQSYEDPWTFGGGTKLEIK antibody clone M14 17EVQLVESGPSLVKPGGSLRLTCSVTGDSITSGYWNWI Caninized heavy chainRKFPGNKLEYMGYISYSGITDYNPSLKSRITISRDTS sequence from mouse antibodyKNQYYLQLNSVTTEDTATYYCARYGNYGYAMDYWGQG clone M14 and canine IgG-ATLVTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLHSLSSMVTVPSSRWPSETFTCNVVHPASNTKVDKPVFNECRCTDTPCPVPEPLGGPSVLIFPPKPKDILRITRIPEVTCVVLDLGREDPEVQISWFVDGKEVHTAKTQSREQQFNGTYRVVSVLPIEHQDWLTGKEFKCRVNHIDLPSPIERTISKARGRAHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVMHETLQNHYTDLSLSH SPGK 18EVQLVESGPSLVKPGGSLRLTCSVTGDSITSGYWNWI Caninized heavy chainRKFPGNKLEYMGYISYSGITDYNPSLKSRITISRDTS sequence from mouse antibodyKNQYYLQLNSVTTEDTATYYCARYGNYGYAMDYWGQG clone M14 and canine IgG-BTLVTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQ ESLSHSPGK 19EVQLVESGPSLVKPGGSLRLTCSVTGDSITSGYWNWI Caninized heavy chainRKFPGNKLEYMGYISYSGITDYNPSLKSRITISRDTS sequence from mouse antibodyKNQYYLQLNSVTTEDTATYYCARYGNYGYAMDYWGQG clone M14 and canine IgG-CTLVTVSSASTTAPSVFPLAPSCGSQSGSTVALACLVSGYIPEPVTVSWNSVSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETFTCNVAHPATNTKVDKPVAKECECKCNCNNCPCPGCGLLGGPSVFIFPPKPKDILVTARTPTVTCVVVDLDPENPEVQISWFVDSKQVQTANTQPREEQSNGTYRVVSVLPIGHQDWLSGKQFKCKVNNKALPSPIEEIISKTPGQAHQPNVYVLPPSRDEMSKNTVTLTCLVKDFFPPEIDVEWQSNGQQEPESKYRMTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQIS LSHSPGK 20EVQLVESGPSLVKPGGSLRLTCSVTGDSITSGYWNWI Caninized heavy chainRKFPGNKLEYMGYISYSGITDYNPSLKSRITISRDTS sequence from mouse antibodyKNQYYLQLNSVTTEDTATYYCARYGNYGYAMDYWGQG clone M14 and canine IgG-DTLVTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSTVTVPSSRWPSETFTCNVVHPASNTKVDKPVPKESTCKCISPCPVPESLGGPSVFIFPPKPKDILRITRIPEITCVVLDLGREDPEVQISWFVDGKEVHTAKTQPREQQFNSTYRVVSVLPIEHQDWLTGKEFKCRVNHIGLPSPIERTISKARGQAHQPSVYVLPPSPKELSSSDTVTLICLIKDFFPPEIDVEWQSNGQPEPESKYHTTAPQLDEDGSYFLYSKLSVDKSRWQQGDTFTCAVMHEALQNHYTDLSLS HSPGK 21DIVMTQSPASLSVSLGQRATISCRASESVDTYGNSFM Caninized light chain sequenceHWYQQKPGQSPKLLIYRASNLESGIPARFGGSGSGTD from mouse antibody cloneFTLTIDPVQADDVATYYCQQSYEDPWTFGGGTKLEIK M14 and canine light chainRNDAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDI constant regionNVKWKVDGVIQDTGIQESVTEQDKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSECQRVD 22MLSHTGPSRFALFLLCSMETLLSSHMAPTHQLPPSDV Canine IL31 amino acidRKIILELQPLSRGLLEDYQKKETGVPESNRTLLLCLT sequenceSDSQPPRLNSSAILPYFRAIRPLSDKNIIDKIIEQLDKLKFQHEPETEISVPADTFECKSFILTILQQFSACLE SVFKSLNSGPQ 23 PSDVRKIILELQPLSRGCanine IL31 epitope 24 DIVLTQSPASLAVSLGQRATISCRASESVDTYGNSFMVariable light chain amino HWYQQKSGQSPKLLIYRASNLESGIPARFGGSGSRTDacid sequence of mouse FTLTIDPVEADDVATYYCQQSYEDPWTFGGGTKLEIKantibody clone M14 25 EVQLQESGPSLVKPSQTLSLTCSVTGDSITSGYWMWIVariable heavy chain amino RKFPGNKLEYMGYISYSGITDYNPSLKSRISITRDTSacid sequence of mouse KNQYYLQLNSVTTEDTATYYCARYGNYGYAMDYWGQGantibody clone M14 TSVTVSS 26 DIVLTQSPASLAVSLGQRATISCRASESVDTYGNSFMChimeric variable light chain HWYQQKSGQSPKLLIYRASNLESGIPARFGGSGSRTDof mouse antibody clone M14 FTLTIDPVEADDVATYYCQQSYEDPWTFGGGTKLEIKand canine light chain constant RNDAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDIregion NVKWKVDGVIQDTGIQESVTEQDKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSECQRVD 27EVQLQESGPSLVKPSQTLSLTCSVTGDSITSGYWNWI Chimeric variable heavy chainRKFPGNKLEYMGYISYSGITDYNPSLKSRISITRDTS of mouse antibody clone M14KNQYYLQLNSVTTEDTATYYCARYGNYGYAMDYWGQG and canine IgG-BTSVTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQ ESLSHSPGK 28MLSHAGPARFALFLLCCMETLLPSHMAPAHRLQPSDV Feline IL31 amino acidRKIILELRPMSKGLLQDYLKKEIGLPESNHSSLPCLS sequenceSDSQLPHINGSAILPYFRAIRPLSDKNTIDKIIEQLDKLKFQREPEAKVSMPADNFERKNFILAVLQQFSACLE HVLQSLNSGPQ 29MVSHIGSTRFALFLLCCLGTLMFSHTGPIYQLQPKEI Equine IL31 amino acidQAIIVELQNLSKKLLDDYLNKEKGVQKFDSDLPSCFT sequenceSDSQAPGNINSSAILPYFKAISPSLNNDKSLYIIEQLDKLNFQNAPETEVSMPTDNFERKRFILTILRWFSNCLELAMKTLTTAEQALPPLDPSTPHAGAVALTHHQQDRT ALDRAVFPFVWAAPRGGEVGDGGH 30DIVLTQSPASLAVSLGQRATISCRASESVDTYGNSFM Chimeric variable light chainHWYQQKSGQSPKLLIYRASNLESGIPARFGGSGSRTD of mouse antibody clone M14FTLTIDPVEADDVATYYCQQSYEDPWTFGGGTKLEIK and feline light chain constantRSDAQPSVFLFQPSLDELHTGSASIVCILNDFYPKEV regionNVKWKVDGVVQNKGIQESTTEQNSKDSTYSLSSTLTMSSTEYQSHEKFSCEVTHKSLASTLVKSFNRSECQRE 31EVQLQESGPSLVKPSQTLSLTCSVTGDSITSGYWNWI Chimeric variable heavy chainRKFPGNKLEYMGYISYSGITDYNPSLKSRISITRDTS of mouse antibody clone M14KNQYYLQLNSVTTEDTATYYCARYGNYGYAMDYWGQG and feline heavy chainTSVTVSSASTTAPSVFPLAPSCGTTSGATVALACLVL constant regionGYFPEPVTVSWNSGALTSGVHTFPAVLQASGLYSLSSMVTVPSSRWLSDTFTCNVAHPPSNTKVDKTVRKTDHPPGPKPCDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKAKGQPHEPQVYVLPPAQEELSRNKVSVTCLIKSFHPPDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFVYSKLSVDRSHWQRGNTYTCSVSHEALHSHHTQ KSLTQSPGK 32EIQMTQSPSSLSASPGDRVTISCRASESVDTYGNSFM Felinized variable light chainHWYQQKPGQSPKLLIYRASNLESGVPSRFSGSGSGTD sequence from mouse antibodyFTLTISSLEPEDAATYYCQQSYEDPWTFGGGTKLEIK clone M14 33DVQLVESGGDLVKPGGSLRLTCSVTGDSITSGYWNWV Felinized variable heavy chainRQAPGKGLQWVAYISYSGITDYADSVKGRFTISRDNA sequence from mouse antibodyKNTLYLQLNNLKAEDTATYYCARYGNYGYAMDYWGQG clone M14 TLVTVSS 34EIQMTQSPSSLSASPGDRVTISCRASESVDTYGNSFM Felinized variable light chainHWYQQKPGQSPKLLIYRASNLESGVPSRFSGSGSGTD sequence from mouse antibodyFTLTISSLEPEDAATYYCQQSYEDPWTFGGGTKLEIK clone M14RSDAQPSVFLFQPSLDELHTGSASIVCILNDFYPKEVNVKWKVDGVVQNKGIQESTTEQNSKDSTYSLSSTLTMSSTEYQSHEKFSCEVTHKSLASTLVKSFNRSECQRE 35DVQLVESGGDLVKPGGSLRLTCSVTGDSITSGYWNWV Felinized variable heavy chainRQAPGKGLQWVAYISYSGITDYADSVKGRFTISRDNA sequence from mouse antibodyKNTLYLQLNNLKAEDTATYYCARYGNYGYAMDYWGQG clone M14TLVTVSSASTTAPSVFPLAPSCGTTSGATVALACLVLGYFPEPVTVSWNSGALTSGVHTFPAVLQASGLYSLSSMVTVPSSRWLSDTFTCNVAHPPSNTKVDKTVRKTDHPPGPKPCDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKAKGQPHEPQVYVLPPAQEELSRNKVSVTCLIKSFHPPDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFVYSKLSVDRSHWQRGNTYTCSVSHEALHSHHTQ KSLTQSPGK

DESCRIPTION OF CERTAIN EMBODIMENTS

Antibodies that bind canine IL31, feline IL31, or equine IL31 areprovided. Antibody heavy chains and light chains that are capable offorming antibodies that bind IL31 are also provided. In addition,antibodies, heavy chains, and light chains comprising one or moreparticular complementary determining regions (CDRs) are provided.Polynucleotides encoding antibodies to canine IL31 are provided. Methodsof producing or purifying antibodies to canine IL31 are also provided.Methods of treatment using antibodies to canine IL31 are provided. Suchmethods include, but are not limited to, methods of treatingIL31-induced conditions in companion animal species. Methods ofdetecting IL31 in a sample from a companion animal species are provided.

For the convenience of the reader, the following definitions of termsused herein are provided.

As used herein, numerical terms such as Kd are calculated based uponscientific measurements and, thus, are subject to appropriatemeasurement error. In some instances, a numerical term may includenumerical values that are rounded to the nearest significant figure.

As used herein, “a” or “an” means “at least one” or “one or more” unlessotherwise specified. As used herein, the term “or” means “and/or” unlessspecified otherwise. In the context of a multiple dependent claim, theuse of “or” when referring back to other claims refers to those claimsin the alternative only.

Anti-IL31 Antibodies

Novel antibodies directed against IL31 are provided, for exampleantibodies that bind to canine IL31, feline IL31, and equine IL31.Anti-IL31 antibodies provided herein include, but are not limited to,monoclonal antibodies, mouse antibodies, chimeric antibodies, caninizedantibodies, felinized antibodies, and equinized antibodies. In someembodiments, an anti-IL31 antibody is an isolated mouse monoclonalantibody such as M14, M18, M19, and M87.

Monoclonal antibodies M14, M18, M19, and M87 were isolated as follows.Briefly, mice were immunized with canine IL31 and mouse monoclonalantibody clones were obtained through standard hybridoma technology.Enzyme linked immunosorbent assay (ELISA) was used to screen forhybridoma clones producing IL31-binding antibodies. Based on bindingaffinity and a cell-based functional assay described herein, hybridomaclones producing monoclonal antibodies M14, M18, M19, and M87 wereselected for further investigation. The variable heavy chain (VH) andvariable light chain (VL) of each of the four clones were sequenced andanalyzed by sequence alignment (FIG. 1).

Also provided herein are amino acid sequences of monoclonal antibodyM14. For example, the variable heavy chain CDRs (SEQ ID NOs: 1-3),variable light chain CDRs (SEQ ID NOs: 8-10), variable region heavychain framework sequences (SEQ ID NOs: 4-7), and variable region lightchain framework sequences (SEQ ID NOs: 11-14) for monoclonal antibodyM14 are provided. The amino acid sequences of the variable light chainand variable heavy chain of monoclonal antibody M14 are provided (SEQ IDNOs: 24 and 25, respectively). In addition, the amino acid sequences ofthe CDRs, framework sequences, variable light chain, variable heavychain of monoclonal antibodies M18, M19, and M87 are provided (FIG. 1).

Also provided herein are chimeric, caninized, felinized, and equinizedantibodies derived from monoclonal antibody M14. In some embodiments,amino acid sequences of caninized monoclonal antibody M14 are provided,such as SEQ ID NOs: 15-21. In some embodiments, amino acid sequences offelinized antibodies derived from monoclonal antibody M14 are provided,such as SEQ ID NOs: 32-35. In some embodiments, amino acid sequences ofchimeric antibodies derived from monoclonal antibody M14 are provided,such as SEQ ID NOs: 26, 27, 30, and 31.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (forexample, bispecific (such as Bi-specific T-cell engagers) andtrispecific antibodies), and antibody fragments (such as Fab, F(ab′)2,ScFv, minibody, diabody, triabody, and tetrabody) so long as theyexhibit the desired antigen-binding activity. Canine, feline, and equinespecies have different varieties (classes) of antibodies that are sharedby many mammalians.

The term antibody includes, but is not limited to, fragments that arecapable of binding to an antigen, such as Fv, single-chain Fv (scFv),Fab, Fab′, di-scFv, sdAb (single domain antibody) and (Fab′)2 (includinga chemically linked F(ab′)2). Papain digestion of antibodies producestwo identical antigen-binding fragments, called “Fab” fragments, eachwith a single antigen-binding site, and a residual “Fc” fragment, whosename reflects its ability to crystallize readily. Pepsin treatmentyields an F(ab′)2 fragment that has two antigen combining sites and isstill capable of cross-linking antigen. The term antibody also includes,but is not limited to, chimeric antibodies, humanized antibodies, andantibodies of various species such as mouse, human, cynomolgus monkey,canine, feline, equine, etc. Furthermore, for all antibody constructsprovided herein, variants having the sequences from other organisms arealso contemplated. Thus, if a murine version of an antibody isdisclosed, one of skill in the art will appreciate how to transform themurine sequence based antibody into a cat, dog, horse, etc. sequence.Antibody fragments also include either orientation of single chainscFvs, tandem di-scFv, diabodies, tandem tri-sdcFv, minibodies, etc.Antibody fragments also include nanobodies (sdAb, an antibody having asingle, monomeric domain, such as a pair of variable domains of heavychains, without a light chain). An antibody fragment can be referred toas being a specific species in some embodiments (for example, mouse scFvor a canine scFv). This denotes the sequences of at least part of thenon-CDR regions, rather than the source of the construct. In someembodiments, the antibodies comprise a label or are conjugated to asecond moiety.

The terms “label” and “detectable label” mean a moiety attached to anantibody or its analyte to render a reaction (for example, binding)between the members of the specific binding pair, detectable. Thelabeled member of the specific binding pair is referred to as“detectably labeled.” Thus, the term “labeled binding protein” refers toa protein with a label incorporated that provides for the identificationof the binding protein. In some embodiments, the label is a detectablemarker that can produce a signal that is detectable by visual orinstrumental means, for example, incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotinyl moieties that can bedetected by marked avidin (for example, streptavidin containing afluorescent marker or enzymatic activity that can be detected by opticalor colorimetric methods). Examples of labels for polypeptides include,but are not limited to, the following: radioisotopes or radionuclides(for example, ³H, ¹⁴C, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, or¹⁵³Sm); chromogens, fluorescent labels (for example, FITC, rhodamine,lanthanide phosphors), enzymatic labels (for example, horseradishperoxidase, luciferase, alkaline phosphatase); chemiluminescent markers;biotinyl groups; predetermined polypeptide epitopes recognized by asecondary reporter (for example, leucine zipper pair sequences, bindingsites for secondary antibodies, metal binding domains, epitope tags);and magnetic agents, such as gadolinium chelates. Representativeexamples of labels commonly employed for immunoassays include moietiesthat produce light, for example, acridinium compounds, and moieties thatproduce fluorescence, for example, fluorescein. In this regard, themoiety itself may not be detectably labeled but may become detectableupon reaction with yet another moiety.

The term “monoclonal antibody” refers to an antibody of a substantiallyhomogeneous population of antibodies, that is, the individual antibodiescomprising the population are identical except for possiblenaturally-occurring mutations that may be present in minor amounts.Monoclonal antibodies are highly specific, being directed against asingle antigenic site. Furthermore, in contrast to poly clonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. Thus, a sample ofmonoclonal antibodies can bind to the same epitope on the antigen. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies may be made bythe hybridoma method first described by Kohler and Milstein, 1975,Nature 256:495, or may be made by recombinant DNA methods such asdescribed in U.S. Pat. No. 4,816,567. The monoclonal antibodies may alsobe isolated from phage libraries generated using the techniquesdescribed in McCafferty et al., 1990, Nature 348:552-554, for example.

In some embodiments, the monoclonal antibody is an isolated mouseantibody selected from clone M14, M18, M19, and M87.

“Amino acid sequence,” means a sequence of amino acids residues in apeptide or protein. The terms “polypeptide” and “protein” are usedinterchangeably to refer to a polymer of amino acid residues, and arenot limited to a minimum length. Such polymers of amino acid residuesmay contain natural or non-natural amino acid residues, and include, butare not limited to, peptides, oligopeptides, dimers, trimers, andmultimers of amino acid residues. Both full-length proteins andfragments thereof are encompassed by the definition. The terms alsoinclude post-expression modifications of the polypeptide, for example,glycosylation, sialylation, acetylation, phosphorylation, and the like.Furthermore, for purposes of the present disclosure, a “polypeptide”refers to a protein which includes modifications, such as deletions,additions, and substitutions (generally conservative in nature), to thenative sequence, as long as the protein maintains the desired activity.These modifications may be deliberate, as through site-directedmutagenesis, or may be accidental, such as through mutations of hostswhich produce the proteins or errors due to PCR amplification.

“IL31” as used herein refers to any native IL31 that results fromexpression and processing of IL31 in a cell. The term includes IL31 fromany vertebrate source, including mammals such as primates (e.g., humansand cynomolgus monkeys) and rodents (e.g., mice and rats), and companionanimals (e.g., dogs, cats, and equine), unless otherwise indicated. Theterm also includes naturally occurring variants of IL31, e.g., splicevariants or allelic variants.

In some embodiments, a canine IL31 comprises the amino acid sequence ofSEQ ID NO: 22. In some embodiments, a feline IL31 comprises the aminoacid sequence of SEQ ID NO: 28. In some embodiments, an equine IL31comprises the amino acid sequence of SEQ ID NO: 29.

The term “IL31 binding domain” of an antibody means the binding domainformed by a light chain and heavy chain of an anti-IL31 antibody, whichbinds IL31.

In some embodiments, the IL31 binding domain binds canine IL31 withgreater affinity than it binds human IL31. In some embodiments, the IL31binding domain binds IL31 of other companion animals, such as felineIL31 or equine IL31.

As used herein, the term “epitope” refers to a site on a target molecule(for example, an antigen, such as a protein, nucleic acid, carbohydrateor lipid) to which an antigen-binding molecule (for example, anantibody, antibody fragment, or scaffold protein containing antibodybinding regions) binds. Epitopes often include a chemically activesurface grouping of molecules such as amino acids, polypeptides or sugarside chains and have specific three dimensional structuralcharacteristics as well as specific charge characteristics. Epitopes canbe formed both from contiguous or juxtaposed noncontiguous residues (forexample, amino acids, nucleotides, sugars, lipid moiety) of the targetmolecule. Epitopes formed from contiguous residues (for example, aminoacids, nucleotides, sugars, lipid moiety) typically are retained onexposure to denaturing solvents whereas epitopes formed by tertiaryfolding typically are lost on treatment with denaturing solvents. Anepitope may include but is not limited to at least 3, at least 5 or 8-10residues (for example, amino acids or nucleotides). In some examples anepitope is less than 20 residues (for example, amino acids ornucleotides) in length, less than 15 residues or less than 12 residues.Two antibodies may bind the same epitope within an antigen if theyexhibit competitive binding for the antigen. In some embodiments, anepitope can be identified by a certain minimal distance to a CDR residueon the antigen-binding molecule. In some embodiments, an epitope can beidentified by the above distance, and further limited to those residuesinvolved in a bond (for example, a hydrogen bond) between an antibodyresidue and an antigen residue. An epitope can be identified by variousscans as well, for example an alanine or arginine scan can indicate oneor more residues that the antigen-binding molecule can interact with.Unless explicitly denoted, a set of residues as an epitope does notexclude other residues from being part of the epitope for a particularantibody. Rather, the presence of such a set designates a minimal series(or set of species) of epitopes. Thus, in some embodiments, a set ofresidues identified as an epitope designates a minimal epitope ofrelevance for the antigen, rather than an exclusive list of residues foran epitope on an antigen.

In some embodiments, the epitope comprises the amino acid sequence ofSEQ ID NO: 23. In some embodiments, the epitope is within amino acids34-50 of SEQ ID NO: 22. In some embodiments, the epitope comprises aminoacids 34-50 of SEQ ID NO: 22.

The term “CDR” means a complementarity determining region as defined byat least one manner of identification to one of skill in the art. Insome embodiments, CDRs can be defined in accordance with any of theChothia numbering schemes, the Kabat numbering scheme, a combination ofKabat and Chothia, the AbM definition, the contact definition, or acombination of the Kabat, Chothia, AbM, or contact definitions. Thevarious CDRs within an antibody can be designated by their appropriatenumber and chain type, including, without limitation as CDR-H1, CDR-H2,CDR-H3, CDR-L1, CDR-L2, and CDR-L3. The term “CDR” is used herein toalso encompass a “hypervariable region” or HVR, including hypervariableloops.

In some embodiments, an anti-IL31 antibody comprises a heavy chaincomprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:1; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; or(c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3. In someembodiments, an anti-IL31 antibody comprises a light chain comprising(a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8; (b) aCDR-L2 comprising the amino acid sequence of SEQ ID NO: 9; or (c) aCDR-L3 comprising the amino acid sequence of SEQ ID NO: 10.

In some embodiments, an anti-IL31 antibody comprises a heavy chaincomprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:1, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and(c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and alight chain comprising (a) a CDR-L1 comprising the amino acid sequenceof SEQ ID NO: 8, (b) a CDR-L2 comprising the amino acid sequence of SEQID NO: 9, and (c) a CDR-L3 comprising the amino acid sequence of SEQ IDNO: 10.

The term “variable region” as used herein refers to a region comprisingat least three CDRs. In some embodiments, the variable region includesthe three CDRs and at least one framework region (“FR”). The terms“heavy chain variable region” or “variable heavy chain” are usedinterchangeably to refer to a region comprising at least three heavychain CDRs. The terms “light chain variable region” or “variable lightchain” are used interchangeably to refer to a region comprising at leastthree light chain CDRs. In some embodiments, the variable heavy chain orvariable light chain comprises at least one framework region. In someembodiments, an antibody comprises at least one heavy chain frameworkregion selected from HC-FR1, HC-FR2, HC-FR3, and HC-FR4. In someembodiments, an antibody comprises at least one light chain frameworkregion selected from LC-FR1, LC-FR2, LC-FR3, and LC-FR4. The frameworkregions may be juxtaposed between light chain CDRs or between heavychain CDRs. For example, an antibody may comprise a variable heavy chainhaving the following structure:(HC-FR1)-(CDR-H1)-(HC-FR2)-(CDR-H2)-(HC-FR3)-(CDR-H3)-(HC-FR4). Anantibody may comprise a variable heavy chain having the followingstructure: (CDR-H1)-(HC-FR2)-(CDR-H2)-(HC-FR3)-(CDR-H3). An antibody mayalso comprise a variable light chain having the following structure:(LC-FR1)-(CDR-L1)-(LC-FR2)-(CDR-L2)-(LC-FR3)-(CDR-L3)-(LC-FR4). Anantibody may also comprise a variable light chain having the followingstructure: (CDR-L1)-(LC-FR2)-(CDR-L2)-(LC-FR3)-(CDR-L3).

In some embodiments, an anti-IL31 antibody comprises one or more of (a)a variable region heavy chain framework 1 (HC-FR1) sequence of SEQ IDNO: 4, (b) a HC-FR2 sequence of SEQ ID NO: 5, (c) a HC-FR3 sequence ofSEQ ID NO: 6, (d) a HC-FR4 sequence of SEQ ID NO: 7, (e) a variableregion light chain framework 1 (LC-FR1) sequence of SEQ ID NO: 11, (f)an LC-FR2 sequence of SEQ ID NO: 12, (g) an LC-FR3 sequence of SEQ IDNO: 13, or (h) an LC-FR4 sequence of SEQ ID NO: 14. In some embodiments,an anti-IL31 antibody comprises a variable light chain sequence of (a)SEQ ID NO: 16, (b) SEQ ID NO: 24, or (c) SEQ ID NO: 32. In someembodiments, an anti-IL31 antibody comprises a variable heavy chainsequence of (a) SEQ ID NO: 15; (b) SEQ ID NO: 25; or (c) SEQ ID NO: 33.In some embodiments, an anti-IL31 antibody comprises (a) a variablelight chain sequence of SEQ ID NO: 16 and a variable heavy chainsequence of SEQ ID NO: 15; (b) a variable light chain sequence of SEQ IDNO: 24 and a variable heavy chain sequence of SEQ ID NO: 25; or (c) avariable light chain sequence of SEQ ID NO: 32 and a variable heavychain sequence of SEQ ID NO: 33.

The term “constant region” as used herein refers to a region comprisingat least three constant domains. The terms “heavy chain constant region”or “constant heavy chain” are used interchangeably to refer to a regioncomprising at least three heavy chain constant domains, CH1, CH2, andCH3. Nonlimiting exemplary heavy chain constant regions include γ, δ, α,ε, and μ. Each heavy chain constant region corresponds to an antibodyisotype. For example, an antibody comprising a γ constant region is anIgG antibody, an antibody comprising a δ constant region is an IgDantibody, an antibody comprising an α constant region is an IgAantibody, an antibody comprising a μ constant region is an IgM antibody,and an antibody comprising an ε constant region is an IgE antibody.Certain isotypes can be further subdivided into subclasses. For example,IgG antibodies include, but are not limited to, IgG1 (comprising a γ₁constant region), IgG2 (comprising a γ₂ constant region), IgG3(comprising a γ₃ constant region), and IgG4 (comprising a γ₄ constantregion) antibodies; IgA antibodies include, but are not limited to, IgA1(comprising an α₁ constant region) and IgA2 (comprising an α₂ constantregion) antibodies; and IgM antibodies include, but are not limited toIgM1 and IgM2. The terms “light chain constant region” or “constantlight chain” are used interchangeably to refer to a region comprising alight chain constant domain, CL. Nonlimiting exemplary light chainconstant regions include λ and κ. Non-function-altering deletions andalterations within the domains are encompassed within the scope of theterm “constant region” unless designated otherwise. Canine, feline, andequine have antibody classes such as IgG, IgA, IgD, IgE, and IgM. Withinthe canine IgG antibody class are IgG-A, IgG-B, IgG-C, and IgG-D. Withinthe feline IgG antibody class are IgG1a, IgG1b, and IgG2. Within theequine IgG antibody class are IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, andIgG7.

The term “chimeric antibody” or “chimeric” refers to an antibody inwhich a portion of the heavy chain or light chain is derived from aparticular source or species, while at least a part of the remainder ofthe heavy chain or light chain is derived from a different source orspecies. In some embodiments, a chimeric antibody refers to an antibodycomprising at least one variable region from a first species (such asmouse, rat, cynomolgus monkey, etc.) and at least one constant regionfrom a second species (such as human, dog, cat, equine, etc.). In someembodiments, a chimeric antibody comprises at least one mouse variableregion and at least one canine constant region. In some embodiments, achimeric antibody comprises at least one mouse variable region and atleast one feline constant region. In some embodiments, all of thevariable regions of a chimeric antibody are from a first species and allof the constant regions of the chimeric antibody are from a secondspecies. In some embodiments, a chimeric antibody comprises a constantheavy chain region or constant light chain region from a companionanimal. In some embodiments, a chimeric antibody comprises a mousevariable heavy and light chains and a companion animal constant heavyand light chains. For example, a chimeric antibody may comprise a mousevariable heavy and light chains and a canine constant heavy and lightchains; a chimeric antibody may comprise a mouse variable heavy andlight chains and a feline constant heavy and light chains; or a chimericantibody may comprise a mouse variable heavy and light chains and anequine constant heavy and light chains.

In some embodiments, an anti-IL31 antibody comprises a chimeric antibodycomprising:

a. (i) a light chain amino acid sequence of SEQ ID NO: 26; (ii) a heavychain amino acid sequence of SEQ ID NO: 27; or (iii) a light chain aminoacid sequence as in (i) and a heavy chain sequence as in (ii); orb. (i) a light chain amino acid sequence of SEQ ID NO: 30; (ii) a heavychain amino acid sequence of SEQ ID NO: 31; or (iii) a light chain aminoacid sequence as in (i) and a heavy chain sequence as in (ii).

A “canine chimeric” or “canine chimeric antibody” refers to a chimericantibody having at least a portion of a heavy chain or a portion of alight chain derived from a dog. A “feline chimeric” or “feline chimericantibody” refers to a chimeric antibody having at least a portion of aheavy chain or a portion of a light chain derived from a cat. An “equinechimeric” or “equine chimeric antibody” refers to a chimeric antibodyhaving at least a portion of a heavy chain or a portion of a light chainderived from a horse. In some embodiments, a canine chimeric antibodycomprises a mouse variable heavy and light chains and a canine constantheavy and light chains. In some embodiments, a feline chimeric antibodycomprises a mouse variable heavy and light chains and a feline constantheavy and light chains. In some embodiments, an equine chimeric antibodycomprises a mouse variable heavy and light chains and an equine constantheavy and light chains. In some embodiments, the antibody is a chimericantibody comprising murine variable heavy chain framework regions ormurine variable light chain framework regions.

A “canine antibody” as used herein encompasses antibodies produced in acanine; antibodies produced in non-canine animals that comprise canineimmunoglobulin genes or comprise canine immunoglobulin peptides; orantibodies selected using in vitro methods, such as phage display,wherein the antibody repertoire is based on a canine immunoglobulinsequence. The term “canine antibody” denotes the genus of sequences thatare canine sequences. Thus, the term is not designating the process bywhich the antibody was created, but the genus of sequences that arerelevant.

In some embodiments, an anti-IL31 antibody comprises a canine heavychain constant region selected from an IgG-A, IgG-B, IgG-C, and IgG-Dconstant region. In some embodiments, an anti-IL31 antibody is a canineIgG-A, IgG-B, IgG-C, or IgG-D antibody. In some embodiments, ananti-IL31 antibody is (a) a canine IgG-A antibody comprising the heavychain amino acid sequence of SEQ ID NO: 17; (b) a canine IgG-B antibodycomprising the heavy chain amino acid sequence of SEQ ID NO: 18; (c) acanine IgG-C antibody comprising the heavy chain amino acid sequence ofSEQ ID NO: 19; or (d) a canine IgG-D antibody comprising the heavy chainamino acid sequence of SEQ ID NO: 20.

A “feline antibody” as used herein encompasses antibodies produced in afeline; antibodies produced in non-feline animals that comprise felineimmunoglobulin genes or comprise feline immunoglobulin peptides; orantibodies selected using in vitro methods, such as phage display,wherein the antibody repertoire is based on a feline immunoglobulinsequence. The term “feline antibody” denotes the genus of sequences thatare feline sequences. Thus, the term is not designating the process bywhich the antibody was created, but the genus of sequences that arerelevant.

In some embodiments, an anti-IL31 antibody comprises a feline heavychain constant region selected from an IgG1, IgG2a, and IgG2b constantregion. In some embodiments, an anti-IL31 antibody is a feline IgG1,IgG2a, or IgG2b antibody.

An “equine antibody” as used herein encompasses antibodies produced inan equine; antibodies produced in non-equine animals that compriseequine immunoglobulin genes or comprise equine immunoglobulin peptides;or antibodies selected using in vitro methods, such as phage display,wherein the antibody repertoire is based on an equine immunoglobulinsequence. The term “equine antibody” denotes the genus of sequences thatare equine sequences. Thus, the term is not designating the process bywhich the antibody was created, but the genus of sequences that arerelevant.

In some embodiments, an anti-IL31 antibody comprises an equine heavychain constant region selected from an IgG1, IgG2, IgG3, IgG4, IgG5,IgG6 and IgG7 constant region. In some embodiments, an anti-IL31antibody is an equine IgG1, IgG2, IgG3, IgG4, IgG5, IgG6 and IgG7antibody.

A “caninized antibody” means an antibody in which at least one aminoacid in a portion of a non-canine variable region has been replaced withthe corresponding amino acid from a canine variable region. In someembodiments, a caninized antibody comprises at least one canine constantregion (e.g., a γ constant region, an α constant region, a δ constantregion, an ε constant region, a μ constant region, or etc.) or fragmentthereof. In some embodiments, a caninized antibody is an antibodyfragment, such as Fab, scFv, (Fab′)₂, etc. The term “caninized” alsodenotes forms of non-canine (for example, murine) antibodies that arechimeric immunoglobulins, immunoglobulin chains, or fragments thereof(such as Fv, Fab, Fab′, F(ab′)₂ or other antigen-binding sequences ofantibodies) that contain minimal sequence of non-canine immunoglobulin.Caninized antibodies can include canine immunoglobulins (recipientantibody) in which residues from a CDR of the recipient are substitutedby residues from a CDR of a non-canine species (donor antibody) such asmouse, rat, or rabbit having the desired specificity, affinity, andcapacity. In some instances, Fv framework region (FR) residues of thecanine immunoglobulin are replaced by corresponding non-canine residues.Furthermore, the caninized antibody can comprise residues that are foundneither in the recipient antibody nor in the imported CDR or frameworksequences, but are included to further refine and optimize antibodyperformance.

In some embodiments, at least one amino acid residue in a portion of amouse variable heavy chain or a mouse variable light chain has beenreplaced with the corresponding amino acid from a canine variableregion. In some embodiments, the modified chain is fused to a canineconstant heavy chain or a canine constant light chain. In someembodiments, an anti-IL31 antibody is a caninized antibody comprising(a) a heavy chain sequence of SEQ ID NO: 15, (b) a heavy chain sequenceof SEQ ID NO: 17, (c) a heavy chain sequence of SEQ ID NO: 18, (d) aheavy chain sequence of SEQ ID NO: 19, (e) a heavy chain sequence of SEQID NO: 20, (f) a light chain sequence of SEQ ID NO: 16, or (g) a lightchain sequence of SEQ ID NO: 21.

A “felinized antibody” means an antibody in which at least one aminoacid in a portion of a non-feline variable region has been replaced withthe corresponding amino acid from a feline variable region. In someembodiments, a felinized antibody comprises at least one feline constantregion (e.g., a γ constant region, an α constant region, a δ constantregion, an ε constant region, a μ constant region, or etc.) or fragmentthereof. In some embodiments, a felinized antibody is an antibodyfragment, such as Fab, scFv, (Fab′)₂, etc. The term “felinized” alsodenotes forms of non-feline (for example, murine) antibodies that arechimeric immunoglobulins, immunoglobulin chains, or fragments thereof(such as Fv, Fab, Fab′, F(ab′)₂ or other antigen-binding sequences ofantibodies) that contain minimal sequence of non-feline immunoglobulin.Felinized antibodies can include feline immunoglobulins (recipientantibody) in which residues from a CDR of the recipient are substitutedby residues from a CDR of a non-feline species (donor antibody) such asmouse, rat, or rabbit having the desired specificity, affinity, andcapacity. In some instances, Fv framework region (FR) residues of thefeline immunoglobulin are replaced by corresponding non-feline residues.Furthermore, the felinized antibody can comprise residues that are foundneither in the recipient antibody nor in the imported CDR or frameworksequences, but are included to further refine and optimize antibodyperformance.

In some embodiments, at least one amino acid residue in a portion of amouse variable heavy chain or a mouse variable light chain has beenreplaced with the corresponding amino acid from a feline variableregion. In some embodiments, the modified chain is fused to a felineconstant heavy chain or a canine constant light chain. In someembodiments, an anti-IL31 antibody is a felinized antibody comprising(a) a light chain sequence of SEQ ID NO: 32, (b) a light chain sequenceof SEQ ID NO: 34, (c) a heavy chain sequence of SEQ ID NO: 33, or (d) aheavy chain sequence of SEQ ID NO: 35.

An “equinized antibody” means an antibody in which at least one aminoacid in a portion of a non-equine variable region has been replaced withthe corresponding amino acid from an equine variable region. In someembodiments, an equinized antibody comprises at least one equineconstant region (e.g., a γ constant region, an α constant region, a δconstant region, an constant region, a μ constant region, or etc.) orfragment thereof. In some embodiments, an equinized antibody is anantibody fragment, such as Fab, scFv, (Fab′)₂, etc. The term “equinized”also denotes forms of non-equine (for example, murine) antibodies thatare chimeric immunoglobulins, immunoglobulin chains, or fragmentsthereof (such as Fv, Fab, Fab′, F(ab′)₂ or other antigen-bindingsequences of antibodies) that contain minimal sequence of non-equineimmunoglobulin. Equinized antibodies can include equine immunoglobulins(recipient antibody) in which residues from a CDR of the recipient aresubstituted by residues from a CDR of a non-equine species (donorantibody) such as mouse, rat, or rabbit having the desired specificity,affinity, and capacity. In some instances, Fv framework region (FR)residues of the equine immunoglobulin are replaced by correspondingnon-equine residues. Furthermore, the equinized antibody can compriseresidues that are found neither in the recipient antibody nor in theimported CDR or framework sequences, but are included to further refineand optimize antibody performance.

In some embodiments, at least one amino acid residue in a portion of amouse variable heavy chain or a mouse variable light chain has beenreplaced with the corresponding amino acid from an equine variableregion. In some embodiments, the modified chain is fused to an equineconstant heavy chain or a canine constant light chain.

The term “IgX Fc” means the Fc region is derived from a particularantibody isotype (e.g., IgG, IgA, IgD, IgE, IgM, etc.), where “X”denotes the antibody isotype. Thus, “IgG Fc” denotes the Fc region of aγ chain, “IgA Fc” denotes the Fc region of an α chain, “IgD Fc” denotesthe Fc region of a δ chain, “IgE Fc” denotes the Fc region of an cchain, “IgM Fc” denotes the Fc region of a μ chain, etc. In someembodiments, the IgG Fc region comprises CH1, hinge, CH2, CH3, and CL1.“IgX-N-Fc” denotes that the Fc region is derived from a particularsubclass of antibody isotype (such as canine IgG subclass A, B, C, or D;feline IgG subclass 1, 2a, or 2b; or equine IgG subclass IgG1, IgG2,IgG3, IgG4, IgG5, IgG6, or IgG7, etc.), where “N” denotes the subclass.In some embodiments, IgX Fc or IgX-N-Fc regions are derived from acompanion animal, such as a dog, a cat, or a horse. In some embodiments,IgG Fc regions are isolated from canine γ heavy chains, such as IgG-A,IgG-B, IgG-C, or IgG-D. In some instances, IgG Fc regions are isolatedfrom feline γ heavy chains, such as IgG1, IgG2a, or IgG2b. Antibodiescomprising an Fc region of IgG-A, IgG-B, IgG-C, or IgG-D may provide forhigher expression levels in recombination production systems.

The term “affinity” means the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (for example,an antibody) and its binding partner (for example, an antigen). Theaffinity of a molecule X for its partner Y can generally be representedby the dissociation constant (K_(D)). Affinity can be measured by commonmethods known in the art, such as, for example, immunoblot, ELISA KD,KinEx A, biolayer interferometry (BLI), or surface plasmon resonancedevices.

The terms “K_(D),” “K_(d),” “Kd” or “Kd value” as used interchangeablyto refer to the equilibrium dissociation constant of an antibody-antigeninteraction. In some embodiments, the K_(d) of the antibody is measuredby using biolayer interferometry assays using a biosensor, such as anOctet® System (Pall ForteBio LLC, Fremont, Calif.) according to thesupplier's instructions. Briefly, biotinylated antigen is bound to thesensor tip and the association of antibody is monitored for ninetyseconds and the dissociation is monitored for 600 seconds. The bufferfor dilutions and binding steps is 20 mM phosphate, 150 mM NaCl, pH 7.2.A buffer only blank curve is subtracted to correct for any drift. Thedata are fit to a 2:1 binding model using ForteBio data analysissoftware to determine association rate constant (k_(on)), dissociationrate constant (k_(off)), and the K_(d). The equilibrium dissociationconstant (K_(d)) is calculated as the ratio of k_(off)/k_(on). The term“kon” refers to the rate constant for association of an antibody to anantigen and the term “koff” refers to the rate constant for dissociationof an antibody from the antibody/antigen complex.

The term “binds” to an antigen or epitope is a term that is wellunderstood in the art, and methods to determine such binding are alsowell known in the art. A molecule is said to exhibit “binding” if itreacts, associates with, or has affinity for a particular cell orsubstance and the reaction, association, or affinity is detectable byone or more methods known in the art, such as, for example, immunoblot,ELISA KD, KinEx A, biolayer interferometry (BLI), surface plasmonresonance devices, or etc.

“Surface plasmon resonance” denotes an optical phenomenon that allowsfor the analysis of real-time biospecific interactions by detection ofalterations in protein concentrations within a biosensor matrix, forexample using the BIAcore™ system (BIAcore International AB, a GEHealthcare company, Uppsala, Sweden and Piscataway, N.J.). For furtherdescriptions, see Jonsson et al. (1993) Ann. Biol. Clin. 51: 19-26.

“Biolayer interferometry” refers to an optical analytical technique thatanalyzes the interference pattern of light reflected from a layer ofimmobilized protein on a biosensor tip and an internal reference layer.Changes in the number of molecules bound to the biosensor tip causeshifts in the interference pattern that can be measured in real-time. Anonlimiting exemplary device for biolayer interferometry is an Octet®system (Pall ForteBio LLC). See, e.g., Abdiche et al., 2008, Anal.Biochem. 377: 209-277.

In some embodiments, an anti-IL31 antibody binds to canine IL31, felineIL31, or equine IL31 with a dissociation constant (Kd) of less than5×10⁻⁶ M, less than 1×10⁻⁶ M, less than 5×10⁻⁷M, less than 1×10⁻⁷M, lessthan 5×10⁻⁸M, less than 1×10⁻⁸M, less than 5×10⁻⁹ M, less than 1×10⁻⁹ M,less than 5×10⁻¹⁰ less than 1×10⁻¹⁰ M, less than 5×10⁻¹¹ M, less M, than1×10⁻¹¹ M, less than 5×10⁻¹² M, or less than 1×10⁻¹² M, as measured bybiolayer interferometry. In some embodiments, an anti-IL31 antibodybinds to canine IL31, feline IL31, or equine IL31 with a Kd of between5×10⁻⁶ M and 1×10⁻⁶ M, between 5×10⁻⁶ M and 5×10⁻⁷M, between 5×10⁻⁷ Mand 1×10⁻⁷ M, between 5×10⁻⁶ M and 5×10⁻⁸ M, 5×10⁻⁶ M and 1×10⁻⁸ M,between 5×10⁻⁶ M and 5×10⁻⁹ M, between 5×10⁻⁶ M and 1×10⁻⁹ M, between5×10⁻⁶ M and 5×10⁻¹⁰ M, between 5×10⁻⁶ M and 1×10⁻¹⁰ M, between 5×10⁻⁶ Mand 5×10⁻¹¹M, between 5×10⁻⁶ M and 1×10⁻¹¹ M, between 5×10⁻⁶ M and5×10⁻¹²M, between 5×10⁻⁶ M and 1×10⁻¹² M, between 1×10⁻⁶ M and 5×10⁻⁷ M,between 1×10⁻⁶ M and 1×10⁻⁷ M, between 1×10⁻⁶ M and 5×10⁻⁸M, 1×10⁻⁶ Mand 1×10⁻⁸M, between 1×10⁻⁶ M and 5×10⁻⁹M, between 1×10⁻⁶ M and 1×10⁻⁹M, between 1×10⁻⁶ M and 5×10⁻¹⁰ M, between 1×10⁻⁶ M and 1×10⁻¹⁰ M,between 1×10⁻⁶M and 5×10⁻¹¹M, between 1×10⁻⁶M and 1×10⁻¹¹M, between1×10⁻⁶M and 5×10⁻¹² M, between 1×10⁻⁶ M and 1×10⁻¹²M, between 5×10⁻⁷ Mand 1×10⁻⁷ M, between 5×10⁻⁷M and 5×10⁻⁸M, 5×10⁻⁷M and 1×10⁻⁸M, between5×10⁻⁷M and 5×10⁻⁹M, between 5×10⁻⁷ M and 1×10⁻⁹ M, between 5×10⁻⁷M and5×10⁻¹⁰ M, between 5×10⁻⁷ M and 1×10⁻¹⁰ M, between 5×10⁻⁷M and 5×10⁻¹¹M, between 5×10⁻⁷ M and 1×10⁻¹¹M, between 5×10⁻⁷M and 5×10⁻¹²M, between5×10⁻⁷M and 1×10⁻¹²M, between 1×10⁻⁷ M and 5×10⁻⁸ M, 1×10⁻⁷ M and 1×10⁻⁸M, between 1×10⁻⁷ M and 5×10⁻⁹ M, between 1×10⁻⁷ M and 1×10⁻⁹ M, between1×10⁻⁷M and 5×10⁻¹⁰ M, between 1×10⁻⁷M and 1×10⁻¹⁰ M, between 1×10⁻⁷Mand 5×10⁻¹¹ M, between 1×10⁻⁷M and 1×10⁻¹¹ M, between 1×10⁻⁷M and5×10⁻¹² M, between 1×10⁻⁷M and 1×10¹²M, between 5×10⁻⁸M and 1×10⁻⁸M,between 5×10⁻⁸M and 5×10⁻⁹M, between 5×10⁻⁸ M and 1×10⁻⁹ M, between5×10⁻⁸M and 5×10⁻¹⁰ M, between 5×10⁻⁸M and 1×10⁻¹⁰ M, between 5×10⁻⁸Mand 5×10⁻¹¹ M, between 5×10⁻⁸M and 1×10⁻¹¹ M, between 5×10⁻⁸M and5×10⁻¹² M, between 5×10⁻⁸M and 1×10⁻¹² M, 1×10⁻⁸M and 5×10⁻⁹M, between1×10⁻⁸M and 1×10⁻⁹ M, between 1×10⁻⁸M and 5×10⁻¹⁰ M, between 1×10⁻⁸ Mand 1×10⁻¹⁰ M, between 1×10⁻⁸M and 5×10⁻¹¹ M, between 1×10⁻⁸ M and1×10⁻¹¹M, between 1×10⁻⁸M and 5×10¹²M, between 1×10⁻⁸M and 1×10¹²M,between 5×10⁻⁹M and 1×10⁻⁹M, between 5×10⁻⁹ M and 5×10⁻¹⁰ M, between5×10⁻⁹ M and 1×10⁻¹⁰ M, between 5×10⁻⁹ M and 5×10⁻¹¹ M, between 5×10⁻⁹ Mand 1×10⁻¹¹ M, between 5×10⁻⁹M and 5×10⁻¹² M, between 5×10⁻⁹ M and1×10⁻¹² M, between 1×10⁻⁹ M and 5×10⁻¹⁰ M, between 1×10⁻⁹ M and 1×10⁻¹⁰M, between 1×10⁻⁹M and 5×10⁻¹¹M, between 1×10⁻⁹M and 1×10⁻¹¹M, between1×10⁻⁹M and 5×10⁻¹²M, between 1×10⁻⁹M and 1×10⁻¹² M, between 5×10⁻¹⁰ Mand 1×10⁻¹⁰ M, between 5×10⁻¹⁰ M and 5×10⁻¹¹ M, between, 1×10⁻¹⁰ M and5×10⁻¹¹ M, 1×10⁻¹⁰ M and 1×10⁻¹¹ M, between 1×10⁻¹⁰ M and 5×10⁻¹² M,between 1×10⁻¹⁰ M and 1×10⁻¹² M, between 5×10⁻¹¹ M and 1×10⁻¹² M,between 5×10⁻¹¹ M and 5×10⁻¹² M, between 5×10⁻¹¹ M and 1×10⁻¹² M,between 1×10⁻¹¹ M and 5×10⁻¹² M, or between 1×10⁻¹¹ M and 1×10⁻¹² M, asmeasured by biolayer interferometry. In some embodiments, an anti-IL31antibody binds to canine IL31, feline IL31, or equine IL31, asdetermined by immunoblot analysis.

In some embodiments, an anti-IL31 antibody is provided that competeswith an anti-IL31 antibody described herein (such as M14, M18, M19, orM87) for binding to IL31. In some embodiments, an antibody that competeswith binding with any of the antibodies provided herein can be made orused. In some embodiments, an anti-IL31 antibody is provided thatcompetes with monoclonal M14 antibody in binding to canine IL31, felineIL31, or equine IL31.

A “variant” means a biologically active polypeptide having at leastabout 50% amino acid sequence identity with the native sequencepolypeptide after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. Such variants include, for instance, polypeptides wherein oneor more amino acid residues are added, deleted, at the N- or C-terminusof the polypeptide.

In some embodiments, a variant has at least about 50% amino acidsequence identity, at least about 60% amino acid sequence identity, atleast about 65% amino acid sequence identity, at least about 70% aminoacid sequence identity, at least about 75% amino acid sequence identity,at least about 80% amino acid sequence identity, at least about 85%amino acid sequence identity, at least about 90% amino acid sequenceidentity, at least about 95% amino acid sequence identity with thenative sequence polypeptide.

As used herein, “percent (%) amino acid sequence identity” and“homology” with respect to a peptide, polypeptide, or antibody sequenceare defined as the percentage of amino acid residues in a candidatesequence that are identical with the amino acid residues in the specificpeptide or polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN, or MEGALINE™ (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor measuring alignment, including any algorithms needed to achievemaximal alignment over the full length of sequences being compared.

An amino acid substitution may include but is not limited to thereplacement of one amino acid in a polypeptide with another amino acid.Exemplary substitutions are shown in Table 2. Amino acid substitutionsmay be introduced into an antibody of interest and the products screenedfor a desired activity, for example, retained/improved antigen binding,decreased immunogenicity, or improved ADCC or CDC.

TABLE 2 Original Residue Exemplary Substitutions Ala (A) Val; Leu; IleArg (R) Lys; Gln; Asn Asn (N) Gln; His; Asp; Lys; Arg Asp (D) Glu; AsnCys (C) Ser; Ala Gln (Q) Asn; Glu Glu (E) Asp; Gln Gly (G) Ala His (H)Asn; Gln; Lys; Arg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu (L)Norleucine; Ile; Val; Met; Ala; Phe Lys (K) Arg; Gln; Asn Met (M) Leu;Phe; Ile Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Pro (P) Ala Ser (S) ThrThr (T) Val; Ser Trp (W) Tyr; Phe Tyr (Y) Trp; Phe; Thr; Ser Val (V)Ile; Leu; Met; Phe; Ala; Norleucine

Amino acids may be grouped according to common side-chain properties:

-   -   (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;    -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   (3) acidic: Asp, Glu;    -   (4) basic: His, Lys, Arg;    -   (5) residues that influence chain orientation: Gly, Pro;    -   (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes with another class.

In some embodiments, an anti-IL31 antibody comprises a heavy chain and alight chain, wherein:

a. the heavy chain comprises a CDR-H1 sequence having at least 85%sequence identity, at least 90% sequence identity, at least 95% sequenceidentity, or at least 98% sequence identity to the amino acid sequenceof SEQ ID NO: 1; a CDR-H2 sequence having at least 85% sequenceidentity, at least 90% sequence identity, at least 95% sequenceidentity, or at least 98% sequence identity to the amino acid sequenceof SEQ ID NO: 2; and a CDR-H3 sequence having at least 85% sequenceidentity, at least 90% sequence identity, at least 95% sequenceidentity, or at least 98% sequence identity to the amino acid sequenceof SEQ ID NO: 3, andb. the light chain comprises a CDR-L1 sequence having at least 85%sequence identity, at least 90% sequence identity, at least 95% sequenceidentity, or at least 98% sequence identity to the amino acid sequenceof SEQ ID NO: 8; a CDR-L2 sequence having at least 85% sequenceidentity, at least 90% sequence identity, at least 95% sequenceidentity, or at least 98% sequence identity to the amino acid sequenceof SEQ ID NO: 9; and a CDR-L3 sequence having at least 85% sequenceidentity, at least 90% sequence identity, at least 95% sequenceidentity, or at least 98% sequence identity to the amino acid sequenceof SEQ ID NO: 10.

In some embodiments, an anti-IL31 antibody comprises a heavy chain and alight chain, wherein:

a. (i) a variable light chain sequence having at least 85%, at least90%, at least 95%, or at least 98% sequence identity to the amino acidsequence of SEQ ID NO: 24; (ii) a variable heavy chain sequence havingat least 85%, at least 90%, at least 95%, or at least 98% sequenceidentity to the amino acid sequence of SEQ ID NO: 25; or (iii) avariable light chain sequence as in (i) and a variable heavy chainsequence as in (ii); orb. (i) a variable light chain sequence having at least 85%, at least90%, at least 95%, or at least 98% sequence identity to the amino acidsequence of SEQ ID NO: 16; (ii) a variable heavy chain sequence havingat least 85%, at least 90%, at least 95%, or at least 98% sequenceidentity to the amino acid sequence of SEQ ID NO: 15; or (iii) avariable light chain sequence as in (i) and a variable heavy chainsequence as in (ii); orc. (i) a variable light chain sequence having at least 85%, at least90%, at least 95%, or at least 98% sequence identity to the amino acidsequence of SEQ ID NO: 32; (ii) a variable heavy chain sequence havingat least 85%, at least 90%, at least 95%, or at least 98% sequenceidentity to the amino acid sequence of SEQ ID NO: 33; or (iii) avariable light chain sequence as in (i) and a variable heavy chainsequence as in (ii).

The term “vector” is used to describe a polynucleotide that can beengineered to contain a cloned polynucleotide or polynucleotides thatcan be propagated in a host cell. A vector can include one or more ofthe following elements: an origin of replication, one or more regulatorysequences (such as, for example, promoters or enhancers) that regulatethe expression of the polypeptide of interest, or one or more selectablemarker genes (such as, for example, antibiotic resistance genes andgenes that can be used in colorimetric assays, for example,β-galactosidase). The term “expression vector” refers to a vector thatis used to express a polypeptide of interest in a host cell.

A “host cell” refers to a cell that may be or has been a recipient of avector or isolated polynucleotide. Host cells may be prokaryotic cellsor eukaryotic cells. Exemplary eukaryotic cells include mammalian cells,such as primate or non-primate animal cells; fungal cells, such asyeast; plant cells; and insect cells. Nonlimiting exemplary mammaliancells include, but are not limited to, NS0 cells, PER.C6® cells(Crucell), 293 cells, and CHO cells, and their derivatives, such as293-6E, DG44, CHO-S, and CHO-K cells. Host cells include progeny of asingle host cell, and the progeny may not necessarily be completelyidentical (in morphology or in genomic DNA complement) to the originalparent cell due to natural, accidental, or deliberate mutation. A hostcell includes cells transfected in vivo with a polynucleotide(s)encoding an amino acid sequence(s) provided herein.

The term “isolated” as used herein refers to a molecule that has beenseparated from at least some of the components with which it istypically found in nature or produced. For example, a polypeptide isreferred to as “isolated” when it is separated from at least some of thecomponents of the cell in which it was produced. Where a polypeptide issecreted by a cell after expression, physically separating thesupernatant containing the polypeptide from the cell that produced it isconsidered to be “isolating” the polypeptide. Similarly, apolynucleotide is referred to as “isolated” when it is not part of thelarger polynucleotide (such as, for example, genomic DNA ormitochondrial DNA, in the case of a DNA polynucleotide) in which it istypically found in nature, or is separated from at least some of thecomponents of the cell in which it was produced, for example, in thecase of an RNA polynucleotide. Thus, a DNA polynucleotide that iscontained in a vector inside a host cell may be referred to as“isolated.” In some embodiments, the anti-IL31 antibody is purifiedusing chromatography, such as size exclusion chromatography, ionexchange chromatography, protein A column chromatography, hydrophobicinteraction chromatography, and CHT chromatography.

The term “companion animal species” refers to an animal suitable to be acompanion to humans. In some embodiments, a companion animal species isa small mammal, such as a canine, feline, dog, cat, horse, rabbit,ferret, guinea pig, rodent, etc. In some embodiments, a companion animalspecies is a farm animal, such as a horse, cow, pig, etc.

The term “IL31 signaling function” refers to any one of or combinationof the downstream activities that occurs when IL31 binds its receptor orreceptor complex. In some embodiments, the IL31 signaling functioncomprises activation of Janus kinase (Jak) 1 or Jak 2 signalingmolecules. In some embodiments, the IL31 signaling function comprisesphosphorylation of STAT-3 or STAT-5 proteins. In some embodiments, theIL31 signaling function comprises activating the ERK1/2 MAP kinasesignaling pathway. In some embodiments, the IL31 signaling functioncomprises activating the PI3K/AKT signaling pathway. In someembodiments, the IL31 signaling function comprises activating the Jak1/2signaling pathway.

“STAT phosphorylation” means the post-expression modification of a STATprotein by phosphorylation. For example, “STAT-3 phosphorylation” refersto the phosphorylation of STAT-3 and “STAT-5 phosphorylation” refers tothe phosphorylation of STAT-5. In some embodiments, the phosphorylationof STAT-3 is measured by immune-blot analysis. For example, cells (e.g.,canine monocytic DH82 cells) are plated into a 96-well cell cultureplate at a density of 1×10⁵ cells per well in growth media (e.g., MEM,Life Technologies®) containing 15% heat-inactivated fetal bovine serum,2 mmol/L GlutaMax, 1 mmol/L sodium pyruvate, and 10 nm/mL canineinterferon-c (R&D Systems, Minneapolis, Minn., USA) for 24 hours at 37°C. in the presence of anti-IL31 antibody as described herein.Immuno-blot analysis of the cell lysate using anti-phospho STAT-3 andanti-STAT-3 antibodies (R&D Systems) were used to detect theconcentration of phosphorylated STAT-3 and unphosphorylated STAT-3relative to each other and compared to a beta-actin control. Methods fordetermining the concentration of proteins, either qualitatively orquantitatively, by immunoblot are understood by persons of skill in theart. In some embodiments, relative concentration is determined byqualitatively by visual inspection of the immunoblot. In someembodiments, the concentration of phosphorylated STAT-3 andunphosphorylated STAT-3 is quantitatively determined by digitallyimaging an immunoblot, determining the intensity of the bands, and usinga linear standard curve of known concentrations of STAT-3 protein toback calculate the concentration of phosphorylated or unphosporylatedSTAT-3 in a sample.

To “reduce” or “inhibit” means to decrease, reduce, or arrest anactivity, function, or amount as compared to a reference. In someembodiments, by “reduce” or “inhibit” is meant the ability to cause anoverall decrease of 20% or greater. In some embodiments, by “reduce” or“inhibit” is meant the ability to cause an overall decrease of 50% orgreater. In some embodiments, by “reduce” or “inhibit” is meant theability to cause an overall decrease of 75%, 85%, 90%, 95%, or greater.In some embodiments, the amount noted above is inhibited or decreasedover a period of time, relative to a control dose (such as a placebo)over the same period of time. A “reference” as used herein, refers toany sample, standard, or level that is used for comparison purposes. Areference may be obtained from a healthy or non-diseased sample. In someexamples, a reference is obtained from a non-diseased or non-treatedsample of a companion animal. In some examples, a reference is obtainedfrom one or more healthy animals of a particular species, which are notthe animal being tested or treated.

The term “substantially reduced,” as used herein, denotes a sufficientlyhigh degree of reduction between a numeric value and a reference numericvalue such that one of skill in the art would consider the differencebetween the two values to be of statistical significance within thecontext of the biological characteristic measured by said values. Insome embodiments, the substantially reduced numeric values is reduced bygreater than about any one of 10%, 15% 20%, 25%, 30%, 35%, 40%, 45%,50%, 60%, 70%, 80%, 90%, or 100% compared to the reference value.

In some embodiments, an IL31 antibody may reduce IL31 signaling functionin a companion animal species by at least 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, or 100% compared to IL31 signaling function in the absence of theantibody, as measured by a reduction in STAT-3 phosphorylation. In someembodiments, the reduction in IL31 signaling function or the reductionin STAT-3 phosphorylation is between 10% and 15%, between 10% and 20%,between 10% and 25%, between 10% and 30%, between 10% and 35%, between10% and 40%, between 10% and 45%, between 10% and 50%, between 10% and60%, between 10% and 70%, between 10% and 80%, between 10% and 90%,between 10% and 100%, between 15% and 20%, between 15% and 25%, between15% and 30%, between 15% and 35%, between 15% and 40%, between 15% and45%, between 15% and 50%, between 15% and 60%, between 15% and 70%,between 15% and 80%, between 15% and 90%, between 15% and 100%, between20% and 25%, between 20% and 30%, between 20% and 35%, between 20% and40%, between 20% and 45%, between 20% and 50%, between 20% and 60%,between 20% and 70%, between 20% and 80%, between 20% and 90%, between20% and 100%, between 25% and 30%, between 25% and 35%, between 25% and40%, between 25% and 45%, between 25% and 50%, between 25% and 60%,between 25% and 70%, between 25% and 80%, between 25% and 90%, between25% and 100%, between 30% and 35%, between 30% and 40%, between 30% and45%, between 30% and 50%, between 30% and 60%, between 30% and 70%,between 30% and 80%, between 30% and 90%, between 30% and 100%, between35% and 40%, between 35% and 45%, between 35% and 50%, between 35% and60%, between 35% and 70%, between 35% and 80%, between 35% and 90%,between 35% and 100%, between 40% and 45%, between 40% and 50%, between40% and 60%, between 40% and 70%, between 40% and 80%, between 40% and90%, between 40% and 100%, between 45% and 50%, between 45% and 60%,between 45% and 70%, between 45% and 80%, between 45% and 90%, between45% and 100%, between 50% and 60%, between 50% and 70%, between 50% and80%, between 50% and 90%, between 50% and 100%, between 60% and 70%,between 60% and 80%, between 60% and 90%, between 60% and 100%, between70% and 80%, between 70% and 90%, between 70% and 100%, between 80% and90%, between 80% and 100%, or between 90% and 100%.

Pharmaceutical Compositions

The terms “pharmaceutical formulation” and “pharmaceutical composition”refer to a preparation which is in such form as to permit the biologicalactivity of the active ingredient(s) to be effective, and which containsno additional components that are unacceptably toxic to a subject towhich the formulation would be administered.

A “pharmaceutically acceptable carrier” refers to a non-toxic solid,semisolid, or liquid filler, diluent, encapsulating material,formulation auxiliary, or carrier conventional in the art for use with atherapeutic agent that together comprise a “pharmaceutical composition”for administration to a subject. A pharmaceutically acceptable carrieris non-toxic to recipients at the dosages and concentrations employedand is compatible with other ingredients of the formulation. Thepharmaceutically acceptable carrier is appropriate for the formulationemployed. Examples of pharmaceutically acceptable carriers includealumina; aluminum stearate; lecithin; serum proteins, such as humanserum albumin, canine or other animal albumin; buffers such asphosphate, citrate, tromethamine or HEPES buffers; glycine; sorbic acid;potassium sorbate; partial glyceride mixtures of saturated vegetablefatty acids; water; salts or electrolytes, such as protamine sulfate,disodium hydrogen phosphate, potassium hydrogen phosphate, sodiumchloride, zinc salts, colloidal silica, or magnesium trisilicate;polyvinyl pyrrolidone, cellulose-based substances; polyethylene glycol;sucrose; mannitol; or amino acids including, but not limited to,arginine.

The pharmaceutical composition can be stored in lyophilized form. Thus,in some embodiments, the preparation process includes a lyophilizationstep. The lyophilized composition may then be reformulated, typically asan aqueous composition suitable for parenteral administration, prior toadministration to the dog, cat, or horse. In other embodiments,particularly where the antibody is highly stable to thermal andoxidative denaturation, the pharmaceutical composition can be stored asa liquid, i.e., as an aqueous composition, which may be administereddirectly, or with appropriate dilution, to the dog, cat, or horse. Alyophilized composition can be reconstituted with sterile Water forInjection (WFI). Bacteriostatic reagents, such benzyl alcohol, may beincluded. Thus, the invention provides pharmaceutical compositions insolid or liquid form.

The pH of the pharmaceutical compositions may be in the range of fromabout pH 5 to about pH 8, when administered. The compositions of theinvention are sterile if they are to be used for therapeutic purposes.Sterility can be achieved by any of several means known in the art,including by filtration through sterile filtration membranes (e.g., 0.2micron membranes). Sterility may be maintained with or withoutanti-bacterial agents.

Uses of Antibodies and Pharmaceutical Compositions

The antibodies or pharmaceutical compositions comprising the antibodiesof the invention may be useful for treating an IL-31-induced condition.As used herein, an “IL31-induced condition” means a disease associatedwith, caused by, or characterized by, elevated levels or alteredgradients of IL31 concentration. Such IL31-induced conditions include,but are not limited to, a pruritic or an allergic disease. In someembodiments, the IL31-induced condition is atopic dermatitis, pruritus,asthma, psoriasis, scleroderma, or eczema. An IL31-induced condition maybe exhibited in a companion animal, including, but not limited to,canine, feline, or equine.

As used herein, “treatment” is an approach for obtaining beneficial ordesired clinical results. “Treatment” as used herein, covers anyadministration or application of a therapeutic for disease in a mammal,including a companion animal. For purposes of this disclosure,beneficial or desired clinical results include, but are not limited to,any one or more of: alleviation of one or more symptoms, diminishment ofextent of disease, preventing or delaying spread of disease, preventingor delaying recurrence of disease, delay or slowing of diseaseprogression, amelioration of the disease state, inhibiting the diseaseor progression of the disease, inhibiting or slowing the disease or itsprogression, arresting its development, and remission (whether partialor total). Also encompassed by “treatment” is a reduction ofpathological consequence of a proliferative disease. The methodsprovided herein contemplate any one or more of these aspects oftreatment. In-line with the above, the term treatment does not requireone-hundred percent removal of all aspects of the disorder.

In some embodiments, an anti-IL31 antibody or pharmaceuticalcompositions comprising it can be utilized in accordance with themethods herein to treat IL31-induced conditions. In some embodiments, ananti-IL31 antibody or pharmaceutical compositions is administered to acompanion animal, such as a canine, a feline, or equine, to treat anIL31-induced condition.

A “therapeutically effective amount” of a substance/molecule, agonist orantagonist may vary according to factors such as the type of disease tobe treated, the disease state, the severity and course of the disease,the type of therapeutic purpose, any previous therapy, the clinicalhistory, the response to prior treatment, the discretion of theattending veterinarian, age, sex, and weight of the animal, and theability of the substance/molecule, agonist or antagonist to elicit adesired response in the animal. A therapeutically effective amount isalso one in which any toxic or detrimental effects of thesubstance/molecule, agonist or antagonist are outweighed by thetherapeutically beneficial effects. A therapeutically effective amountmay be delivered in one or more administrations. A therapeuticallyeffective amount refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic orprophylactic result.

In some embodiments, an anti-IL31 antibody or pharmaceutical compositioncomprising an anti-IL31 antibody is administered parenterally, bysubcutaneous administration, intravenous infusion, or intramuscularinjection. In some embodiments, an anti-IL31 antibody or pharmaceuticalcomposition comprising an anti-IL31 antibody is administered as a bolusinjection or by continuous infusion over a period of time. In someembodiments, an anti-IL31 antibody or pharmaceutical compositioncomprising an anti-IL31 antibody is administered by an intramuscular, anintraperitoneal, an intracerebrospinal, a subcutaneous, anintra-arterial, an intrasynovial, an intrathecal, or an inhalationroute.

Anti-IL31 antibodies described herein may be administered in an amountin the range of 0.1 mg/kg body weight to 100 mg/kg body weight per dose.In some embodiments, anti-IL31 antibodies may be administered in anamount in the range of 0.5 mg/kg body weight to 50 mg/kg body weight perdose. In some embodiments, anti-IL31 antibodies may be administered inan amount in the range of 1 mg/kg body weight to 10 mg/kg body weightper dose. In some embodiments, anti-IL31 antibodies may be administeredin an amount in the range of 0.5 mg/kg body weight to 100 mg/kg body, inthe range of 1 mg/kg body weight to 100 mg/kg body weight, in the rangeof 5 mg/kg body weight to 100 mg/kg body weight, in the range of 10mg/kg body weight to 100 mg/kg body weight, in the range of 20 mg/kgbody weight to 100 mg/kg body weight, in the range of 50 mg/kg bodyweight to 100 mg/kg body weight, in the range of 1 mg/kg body weight to10 mg/kg body weight, in the range of 5 mg/kg body weight to 10 mg/kgbody weight, in the range of 0.5 mg/kg body weight to 10 mg/kg bodyweight, or in the range of 5 mg/kg body weight to 50 mg/kg body weight.

An anti-IL31 antibody or a pharmaceutical composition comprising ananti-IL31 antibody can be administered to a companion animal at one timeor over a series of treatments. For example, an anti-IL31 antibody or apharmaceutical composition comprising an anti-IL31 antibody may beadministered at least once, more than once, at least twice, at leastthree times, at least four times, or at least five times.

In some embodiments, the dose is administered once per week for at leasttwo or three consecutive weeks, and in some embodiments, this cycle oftreatment is repeated two or more times, optionally interspersed withone or more weeks of no treatment. In other embodiments, thetherapeutically effective dose is administered once per day for two tofive consecutive days, and in some embodiments, this cycle of treatmentis repeated two or more times, optionally interspersed with one or moredays or weeks of no treatment.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive or sequentialadministration in any order. The term “concurrently” is used herein torefer to administration of two or more therapeutic agents, where atleast part of the administration overlaps in time or where theadministration of one therapeutic agent falls within a short period oftime relative to administration of the other therapeutic agent. Forexample, the two or more therapeutic agents are administered with a timeseparation of no more than about a specified number of minutes. The term“sequentially” is used herein to refer to administration of two or moretherapeutic agents where the administration of one or more agent(s)continues after discontinuing the administration of one or more otheragent(s), or wherein administration of one or more agent(s) beginsbefore the administration of one or more other agent(s). For example,administration of the two or more therapeutic agents are administeredwith a time separation of more than about a specified number of minutes.As used herein, “in conjunction with” refers to administration of onetreatment modality in addition to another treatment modality. As such,“in conjunction with” refers to administration of one treatment modalitybefore, during or after administration of the other treatment modalityto the animal.

In some embodiments, the method comprises administering in combinationwith an anti-IL31 antibody or a pharmaceutical composition comprising ananti-IL31 antibody, a Jak inhibitor, a PI3K inhibitor, an AKT inhibitor,or a MAPK inhibitor. In some embodiments, the method comprisesadministering in combination with an anti-IL31 antibody or apharmaceutical composition comprising an anti-IL31 antibody, ananti-IL17 antibody, an anti-TNFα antibody, an anti-CD20 antibody, ananti-CD19 antibody, an anti-CD25 antibody, an anti-IL4 antibody, ananti-IL13 antibody, an anti-IL23 antibody, an anti-IgE antibody, ananti-CD11α antibody, anti-IL6R antibody, anti-α4-Intergrin antibody, ananti-IL12 antibody, an anti-IL1β antibody, or an anti-BlyS antibody.

Provided herein are methods of exposing to a cell an anti-IL31 antibodyor a pharmaceutical composition comprising an anti-IL31 antibody underconditions permissive for binding of the antibody to IL31. In someembodiments, the cell is exposed to the antibody or pharmaceuticalcomposition ex vivo. In some embodiments, the cell is exposed to theantibody or pharmaceutical composition in vivo. In some embodiments, acell is exposed to the anti-IL31 antibody or the pharmaceuticalcomposition under conditions permissive for binding of the antibody tointracellular IL31. In some embodiments, a cell is exposed to theanti-IL31 antibody or the pharmaceutical composition under conditionspermissive for binding of the antibody to extracellular IL31. In someembodiments, a cell may be exposed in vivo to the anti-IL31 antibody orthe pharmaceutical composition by any one or more of the administrationmethods described herein, including but not limited to, intraperitoneal,intramuscular, intravenous injection into the subject. In someembodiments, a cell may be exposed ex vivo to the anti-IL31 antibody orthe pharmaceutical composition by exposing the cell to a culture mediumcomprising the antibody or the pharmaceutical composition. In someembodiments, the permeability of the cell membrane may be affected bythe use of any number of methods understood by those of skill in the art(such as electroporating the cells or exposing the cells to a solutioncontaining calcium chloride) before exposing the cell to a culturemedium comprising the antibody or the pharmaceutical composition.

In some embodiments, the binding results in a reduction of IL31signaling function by the cell. In some embodiments, an IL31 antibodymay reduce IL31 signaling function in a cell by at least 10%, at least15%, at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, or 100% compared to IL31 signaling function in theabsence of the antibody, as measured by a reduction in STAT-3phosphorylation. In some embodiments, the reduction in IL31 signalingfunction or the reduction in STAT-3 phosphorylation is between 10% and15%, between 10% and 20%, between 10% and 25%, between 10% and 30%,between 10% and 35%, between 10% and 40%, between 10% and 45%, between10% and 50%, between 10% and 60%, between 10% and 70%, between 10% and80%, between 10% and 90%, between 10% and 100%, between 15% and 20%,between 15% and 25%, between 15% and 30%, between 15% and 35%, between15% and 40%, between 15% and 45%, between 15% and 50%, between 15% and60%, between 15% and 70%, between 15% and 80%, between 15% and 90%,between 15% and 100%, between 20% and 25%, between 20% and 30%, between20% and 35%, between 20% and 40%, between 20% and 45%, between 20% and50%, between 20% and 60%, between 20% and 70%, between 20% and 80%,between 20% and 90%, between 20% and 100%, between 25% and 30%, between25% and 35%, between 25% and 40%, between 25% and 45%, between 25% and50%, between 25% and 60%, between 25% and 70%, between 25% and 80%,between 25% and 90%, between 25% and 100%, between 30% and 35%, between30% and 40%, between 30% and 45%, between 30% and 50%, between 30% and60%, between 30% and 70%, between 30% and 80%, between 30% and 90%,between 30% and 100%, between 35% and 40%, between 35% and 45%, between35% and 50%, between 35% and 60%, between 35% and 70%, between 35% and80%, between 35% and 90%, between 35% and 100%, between 40% and 45%,between 40% and 50%, between 40% and 60%, between 40% and 70%, between40% and 80%, between 40% and 90%, between 40% and 100%, between 45% and50%, between 45% and 60%, between 45% and 70%, between 45% and 80%,between 45% and 90%, between 45% and 100%, between 50% and 60%, between50% and 70%, between 50% and 80%, between 50% and 90%, between 50% and100%, between 60% and 70%, between 60% and 80%, between 60% and 90%,between 60% and 100%, between 70% and 80%, between 70% and 90%, between70% and 100%, between 80% and 90%, between 80% and 100%, or between 90%and 100%.

Provided herein are methods of using the anti-IL31 antibodies,polypeptides and polynucleotides for detection, diagnosis and monitoringof an IL31-induced condition. Provided herein are methods of determiningwhether a companion animal will respond to anti-IL31 antibody therapy.In some embodiments, the method comprises detecting whether the animalhas cells that express IL31 using an anti-IL31 antibody. In someembodiments, the method of detection comprises contacting the samplewith an antibody, polypeptide, or polynucleotide and determining whetherthe level of binding differs from that of a reference or comparisonsample (such as a control). In some embodiments, the method may beuseful to determine whether the antibodies or polypeptides describedherein are an appropriate treatment for the subject animal.

In some embodiments, the sample is a biological sample. The term“biological sample” means a quantity of a substance from a living thingor formerly living thing. In some embodiments, the biological sample isa cell or cell/tissue lysate. In some embodiments, the biological sampleincludes, but is not limited to, blood, (for example, whole blood),plasma, serum, urine, synovial fluid, and epithelial cells.

In some embodiments, the cells or cell/tissue lysate are contacted withan anti-IL31 antibody and the binding between the antibody and the cellis determined. When the test cells show binding activity as compared toa reference cell of the same tissue type, it may indicate that thesubject would benefit from treatment with an anti-IL31 antibody. In someembodiments, the test cells are from tissue of a companion animal.

Various methods known in the art for detecting specific antibody-antigenbinding can be used. Exemplary immunoassays which can be conductedinclude fluorescence polarization immunoassay (FPIA), fluorescenceimmunoassay (FIA), enzyme immunoassay (EIA), nephelometric inhibitionimmunoassay (NIA), enzyme linked immunosorbent assay (ELISA), andradioimmunoassay (RIA). An indicator moiety, or label group, can beattached to the subject antibodies and is selected so as to meet theneeds of various uses of the method which are often dictated by theavailability of assay equipment and compatible immunoassay procedures.Appropriate labels include, without limitation, radionuclides (forexample ¹²¹I, ¹³¹I, ³⁵S, ³H, or ³²P), enzymes (for example, alkalinephosphatase, horseradish peroxidase, luciferase, or p-glactosidase),fluorescent moieties or proteins (for example, fluorescein, rhodamine,phycoerythrin, GFP, or BFP), or luminescent moieties (for example, Qdot™nanoparticles supplied by the Quantum Dot Corporation, Palo Alto,Calif.). General techniques to be used in performing the variousimmunoassays noted above are known to those of ordinary skill in theart.

For purposes of diagnosis, the polypeptide including antibodies can belabeled with a detectable moiety including but not limited toradioisotopes, fluorescent labels, and various enzyme-substrate labelsknow in the art. Methods of conjugating labels to an antibody are knownin the art. In some embodiments, the anti-IL31 antibodies need not belabeled, and the presence thereof can be detected using a second labeledantibody which binds to the first anti-IL31 antibody. In someembodiments, the anti-IL31 antibody can be employed in any known assaymethod, such as competitive binding assays, direct and indirect sandwichassays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: AManual of Techniques, pp. 147-158 (CRC Press, Inc. 1987). The anti-IL31antibodies and polypeptides can also be used for in vivo diagnosticassays, such as in vivo imaging. Generally, the antibody or thepolypeptide is labeled with a radionuclide (such as ¹¹¹In, ⁹⁹Tc, ¹⁴C,¹³¹I, ¹²⁵I, ³H, or any other radionuclide label, including thoseoutlined herein) so that the cells or tissue of interest can belocalized using immunoscintiography. The antibody may also be used asstaining reagent in pathology using techniques well known in the art.

In some embodiments, a first antibody is used for a diagnostic and asecond antibody is used as a therapeutic. In some embodiments, the firstand second antibodies are different. In some embodiments, the first andsecond antibodies can both bind to the antigen at the same time, bybinding to separate epitopes.

The following examples illustrate particular aspects of the disclosureand are not intended in any way to limit the disclosure.

EXAMPLES Example 1: Identification of Mouse Monoclonal Antibodies thatBind to Canine IL31

Canine IL31 gene encoding IL31 protein (SEQ ID NO: 22) was synthesizedwith poly-His tag on the C-terminal and cloned into a mammalianexpression vector. The plasmid that carries canine IL31 gene wastransfected to 293 cells.

The supernatant containing canine IL31 protein was collected andfiltered. Canine IL31 was affinity purified using Ni-NTA column(CaptivA® Protein A Affinity Resin, Repligen).

Mouse monoclonal antibodies were identified using standard immunizationusing canine IL31 produced by 293 cells as immunogen. Various adjuvantswere used during immunizations (Antibody Solutions, Sunnyvale, Calif.)and monoclonal antibodies were obtained through standard hybridomatechnology. Enzyme linked immunosorbent assay (ELISA) was developed toscreen the clones that produce IL31 binding antibodies. First canineIL31 was biotinylated and then it was introduced to streptavidin-coatedwells. Immunized serum was then added to the wells followed by washingand detection with HRP-conjugated anti-mouse antibodies. The presence ofcanine IL31 binding antibody developed a positive signal. Over 100 ELISApositive clones were rescreened using biosensor (Forte Bio Octet).Biotinylated canine IL31 was bound to the sensor tip and hybridoma clonesupernatants containing anti-canine IL31 antibodies were screened forantibodies having a slow off-rate (the rate of dissociation betweenantibody and ligand). The binding affinity of the top 19 candidates weremeasured at single concentration and reported as the equilibriumdissociation constant (Kd) after the antibody concentrations weremeasured by protein A titer assay using Biosensor Octet. The Kds of the19 candidates were all less than 10 nM.

Furthermore, a cell-based functional assay described below in Example 4,was performed to assess activity of the top candidates in reducingcanine IL31-mediated pSTAT signaling using canine DH82 cells. Four topclones (M14, M18, M19, and M87) were selected for further investigation.

Example 2: Identification of DNA Sequences Encoding VH and VL ofMonoclonal Antibodies

Hybridoma cells producing M14, M18, M19 and M87 were pelleted. RNA wasextracted and oligonucleotide primers for amplifying mouseimmunoglobulin (Ig) variable domains were used to obtain cDNA usingstandard techniques. The variable heavy chain (VH) and variable lightchain (VL) of each of the four clones were sequenced and analyzed bysequence alignment (FIG. 1).

Example 3: Expression and Purification of Murine-Canine Chimeric andCaninized IL31-mAb M14 from CHO Cells

DNA sequences encoding a chimeric antibody were designed for a fusion ofmurine M14 VH (SEQ ID NO: 25) and murine VL (SEQ ID NO: 24) to canineconstant heavy chain and canine constant light chain. The nucleotidesequences were synthesized chemically and inserted into an expressionvector suitable for transfection into a CHO host cell. Aftertransfection into CHO cells, the light chain or heavy chain protein orboth were secreted from the cell. For example, chimeric M14 that usescanine IgG-B was purified by single step Protein A columnchromatography.

Murine M14 VH and VL were caninized by searching and selecting propercanine germline antibody sequences as a template for CDR grafting,followed by protein modeling. Caninized M14 IgG-B (SEQ ID NO: 18 and SEQID NO: 21) was readily expressed and purified in a single step with aprotein A column or other chromatographic methods, such as ion exchangecolumn chromatography, hydrophobic interaction column chromatography,mixed mode column chromatography such as CHT, or multimodal mode columnchromatography such as CaptoMMC. Low pH or other viral inactivation andviral removal steps can be applied. The purified protein is admixed withexcipients, and sterilized by filtration to prepare a pharmaceuticalcomposition of the invention. The pharmaceutical composition isadministered to a dog with an atopic dermatitis in an amount sufficientto bind to inhibit IL31.

The vectors were then used to perform pilot-scale transfection in CHO-Scells using the FreestyleMax™ transfection reagent (Life Technologies).The supernatant was harvested by clarifying the conditioned media.Protein was purified with a single pass Protein A chromatography stepand used for further investigation.

Example 4: Demonstration of IL31 Binding Activity

This example demonstrates that antibodies of the invention, illustratedwith the chimeric M14 (SEQ ID NO:26 and SEQ ID NO:27) and caninized M14(SEQ ID NO:18 and SEQ ID NO:21) bind canine IL31 with kinetics requisitefor therapeutic activity.

The binding analysis was performed using a biosensor Octet as follows.Briefly, canine IL31 was biotinylated. The free unreacted biotin wasremoved from biotinylated IL31 by extensive dialysis. Biotinylatedcanine IL31 was captured on streptavidin sensor tips. The association offour different concentrations (400, 200, 66.6, and 33 nM) of antibodyand IL31 (human and canine, in different tests) was monitored for ninetyseconds. Dissociation was monitored for 600 seconds. A buffer only blankcurve was subtracted to correct for any drift. The data were fit to a2:1 binding model using ForteBio™ data analysis software to determinethe k_(on), k_(off), and the Kd. The buffer for dilutions and allbinding steps was: 20 mM phosphate, 150 mM NaCl, pH 7.2.

Canine IL31 with C-terminal polyHis tag was expressed and purified fromCHO-S cells. Human IL31 was obtained from Sino Biological, EZ-LinkNHS-LC-biotin was obtained from Thermo Scientific (Cat. #21336), andStreptavidin biosensors was obtained from ForteBio (Cat. #18-509).

The binding kinetics were as follows: For the ligand canine IL31, the Kd(M) for chimeric M14 was <1.0×10⁻¹¹ (FIG. 2) and <1.0×10⁻¹¹ (FIG. 3) forcaninized M14.

Chimeric M14 and caninized M14 had no obvious binding signal with humanIL31. Thus the Kd could not be measured.

Example 5: Demonstration that M14 Inhibits Canine IL31 Signaling

After binding to its IL31 receptor, IL-31 activates Janus kinase (Jak) 1and Jak2 signaling molecules. In turn, activated Jaks stimulate thephosphorylation of downstream signaling STAT-3 and STAT-5.Anti-phospho-Stat3 immuno-blot analysis was used to detect anti-IL31activity from a protein A-purified fraction of cell-free culture medium(Gonzales et. al. Vet Dermatol 2013; 24: 48-e12). In Brief, the caninemonocytic DH82 cells (American Type Culture Collection, Manassas, Va.,USA) were plated into 96-well flat-bottomed cell culture plates at adensity of 1×10⁵ cells per well in MEM growth media (Life Technologies)containing 15% heat-inactivated fetal bovine serum, 2 mmol/L GlutaMax, 1mmol/L sodium pyruvate, and 10 ng/mL canine interferon-c (R&D Systems,Minneapolis, Minn., USA) for 24 h at 37° C. In this experiment,concentration of canine IL31-Fc was 5 ng/mL (8 nM). Anti-phospho STAT-3and anti-STAT-3 antibodies were purchased from R&D Systems. Anti-betaactin antibody was from Sigma-Aldrich. As shown in FIG. 4, canine IL31signaling decreased (as evidenced by a reduction in STAT-3phosphorylation) as the concentration of caninized M14 exposed to thecells increased (lane 1: no anti-IL31 antibody; Lane 2: 3.3 nM; Lane 3:6.6 nM; Lane 4: 9.9 nM; and Lane 5: 13.2 nM).

Example 6: Identification of M14 Canine IL31 Binding Epitope

To identify the canine IL31 epitope recognized by M14, multiple GSTcanine IL31 fragment fusion molecules were generated and proteins wereexpressed intracellularly in E. coli. After the GST fusion proteins weretransferred to a membrane, chimeric M14 was used to probe the membrane.A positive signal resulted when the IL31 fragment contained the epitope.

FIG. 5 combined with FIG. 6 demonstrated M14 can recognize the minimalfragment (SEQ ID NO: 23).

Example 7: Demonstrating M14 Cross Reacts to Feline IL31

To examine whether M14 antibody recognizes feline IL31 (SEQ ID NO: 28)or equine IL31 (SEQ ID NO: 29), each protein was fused to human Fc andexpressed in mammalian 293 cells. The partially purified proteins wereblotted to membrane and probed with M14 antibody. The immunoblot of FIG.1 demonstrates that M14 binds to feline IL31. The immunoblot assay didnot detect binding between M14 and equine IL31. However, biolayerinterferometry analysis revealed that M14 antibody binds equine IL31,but with a lesser affinity. The preliminary Kd measurement usingbiotinylated equine IL31 immobilized to the sensor revealed that theaffinity (Kd) is approximately 10 to 50 nM.

Example 8: Felinized M14

M14 variable light chain was felinized as (SEQ ID NO: 32) and M14variable heavy chain was felinized as (SEQ ID NO: 33). First, the mouseheavy chain variable and light chain variable sequences were used tosearch proper variants of feline VH and VL. The proper feline frameswere chosen to graft CDRs. They are further optimized using structuralmodeling. The felinized VH and VL were fused to a feline IgG heavy chainconstant domains (CH1, CH2, and CH3) and feline light chain constantdomain (CL1).

Feline M14 chimeric antibody (SEQ ID NO: 30 and SEQ ID NO: 31) orfelinized M14 antibody (SEQ ID NO: 34 and SEQ ID NO: 35) can be asadministered to cats for treatment of an IL31-induced condition.

1.-25. (canceled)
 26. An isolated nucleic acid encoding an antibody thatbinds to canine IL31, wherein the antibody comprises (a) CDR-H1comprising the amino acid sequence of SEQ ID NO: 1; (b) CDR-H2comprising the amino acid sequence of SEQ ID NO: 2; (c) CDR-H3comprising the amino acid sequence of SEQ ID NO: 3; (d) CDR-L1comprising the amino acid sequence of SEQ ID NO: 8; (e) CDR-L2comprising the amino acid sequence of SEQ ID NO: 9; and (f) CDR-L3comprising the amino acid sequence of SEQ ID NO:
 10. 27. A host cellcomprising the nucleic acid of claim
 26. 28. A method of producing anantibody comprising culturing the host cell of claim 27 and isolatingthe antibody. 29.-43. (canceled)
 44. The isolated nucleic acid of claim26, wherein the antibody binds to canine IL31 with a dissociationconstant (Kd) of less than 5×10⁻¹¹ M, as measured by biolayerinterferometry.
 45. The isolated nucleic acid of claim 26, wherein theantibody is an antibody fragment selected from Fv, scFv, Fab, Fab′,F(ab′)2, and Fab′-SH.
 46. The isolated nucleic acid of claim 26, whereinthe antibody is a caninized, a felinized, or a chimeric antibody. 47.The isolated nucleic acid of claim 26, wherein the antibody furthercomprises one or more of (a) a variable region heavy chain framework 1(HC-FR1) sequence of SEQ ID NO: 4; (b) a HC-FR2 sequence of SEQ ID NO:5; (c) a HC-FR3 sequence of SEQ ID NO: 6; (d) a HC-FR4 sequence of SEQID NO: 7; (e) a variable region light chain framework 1 (LC-FR1)sequence of SEQ ID NO: 11; (f) an LC-FR2 sequence of SEQ ID NO: 12; (g)an LC-FR3 sequence of SEQ ID NO: 13; or (h) an LC-FR4 sequence of SEQ IDNO:
 14. 48. The isolated nucleic acid of claim 26, wherein the antibodycomprises: a) (i) a variable light chain sequence having at least 95%sequence identity to the amino acid sequence of SEQ ID NO: 24; (ii) avariable heavy chain sequence having at least 95% sequence identity tothe amino acid sequence of SEQ ID NO: 25; or (iii) a variable lightchain sequence as in (i) and a variable heavy chain sequence as in (ii);or b) (i) a variable light chain sequence having at least 95% sequenceidentity to the amino acid sequence of SEQ ID NO: 16; (ii) a variableheavy chain sequence having at least 95% sequence identity to the aminoacid sequence of SEQ ID NO: 15; or (iii) a variable light chain sequenceas in (i) and a variable heavy chain sequence as in (ii).
 49. Theisolated nucleic acid of claim 26, wherein the antibody comprises: a)(i) a variable light chain amino acid sequence of SEQ ID NO: 24; (ii) avariable heavy chain amino acid sequence of SEQ ID NO: 25; or (iii) avariable light chain sequence as in (i) and a variable heavy chainsequence as in (ii); or b) (i) a variable light chain amino acidsequence of SEQ ID NO: 16; (ii) a variable heavy chain amino acidsequence of SEQ ID NO: 15; or (iii) a variable light chain sequence asin (i) and a variable heavy chain sequence as in (ii).
 50. The isolatednucleic acid of claim 26, wherein the antibody comprises: a) a canineheavy chain constant region selected from an IgG-A, IgG-B, IgG-C, andIgG-D constant region; or b) a feline heavy chain constant regionselected from an IgG1, IgG2a, and IgG2b constant region.
 51. Theisolated nucleic acid of claim 26, wherein the antibody comprises: a)(i) a light chain amino acid sequence of SEQ ID NO: 26; (ii) a heavychain amino acid sequence of SEQ ID NO: 27; or (iii) a light chainsequence as in (i) and a heavy chain sequence as in (ii); or b) (i) alight chain amino acid sequence of SEQ ID NO: 30; (ii) a heavy chainamino acid sequence of SEQ ID NO: 31; or (iii) a light chain amino acidsequence as in (i) and a heavy chain amino acid sequence as in (ii). 52.The isolated nucleic acid of claim 26, wherein the antibody comprises:(i) a light chain amino acid sequence of SEQ ID NO: 21; (ii) a heavychain amino acid sequence of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:19, or SEQ ID NO: 20; or (iii) a light chain sequence as in (i) and aheavy chain sequence as in (ii).
 53. The isolated nucleic acid of claim26, wherein the antibody comprises: a) a variable light chain sequenceof SEQ ID NO: 24 and a variable heavy chain sequence of SEQ ID NO: 25;or b) a variable light chain sequence of SEQ ID NO: 16 and a variableheavy chain sequence of SEQ ID NO:
 15. 54. The isolated nucleic acid ofclaim 53, wherein the antibody comprises a canine heavy chain constantregion selected from an IgG-A, IgG-B, IgG-C, and IgG-D constant region.55. The isolated nucleic acid of claim 26, wherein the antibodycomprises a light chain amino acid sequence of SEQ ID NO: 21 and a heavychain amino acid sequence of SEQ ID NO:
 17. 56. The isolated nucleicacid of claim 26, wherein the antibody comprises a light chain aminoacid sequence of SEQ ID NO: 21 and a heavy chain amino acid sequence ofSEQ ID NO:
 18. 57. The isolated nucleic acid of claim 26, wherein theantibody comprises a light chain amino acid sequence of SEQ ID NO: 21and a heavy chain amino acid sequence of SEQ ID NO:
 19. 58. The isolatednucleic acid of claim 26, wherein the antibody comprises a light chainamino acid sequence of SEQ ID NO: 21 and a heavy chain amino acidsequence of SEQ ID NO:
 20. 59. An isolated nucleic acid encoding anantibody that binds to canine IL31, wherein the antibody comprises avariable light chain amino acid sequence of SEQ ID NO: 16 and a variableheavy chain amino acid sequence of SEQ ID NO:
 15. 60. An isolatednucleic acid encoding an antibody that binds to canine IL31, wherein theantibody comprises a light chain amino acid sequence of SEQ ID NO: 21and a heavy chain amino acid sequence of SEQ ID NO: 17, SEQ ID NO: 18,SEQ ID NO: 19, or SEQ ID NO: 20.